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Transcriptional and epigenetic mechanisms underlying sensory hair cell differentiation and regeneration

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Transcriptional and epigenetic mechanisms underlying sensory hair cell differentiation and regeneration

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Content Transcriptional and epigenetic
mechanisms underlying
sensory hair cell differentiation
and regeneration

by

Haoze YU

A Dissertation Presented to the
FALCULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(Development, Stem cell and Regenerative Medicine)

December 2020

Copyright 2020 Haoze YU
ii
Dedication

To my fiancée Meng Xing, and our daughter Max.

Life is full of fun, also full of responsibility.

iii
Acknowledgements
I love USC. USC gives me everything. She trained me in doing science, in giving
lectures and reports. She offered me fellowships, honors, opportunities to be
recognized. She helps me keep the qualifications to dream bigger dreams. And
importantly, through USC, I got to know a great mentor, who became the most
important person in my life in the past 5 years. Neil’s influence on me is profound,
especially in thinking and behaving as an independent individual. Maybe I cannot
remember all the jokes and sophisticated expressions coming out of his voice. But there
is one thing I will remember for all my life: “You should do it. It is your life”. When I
asked him for a 5 days leave to hike the north part of the John Muir trail, “You should do
it!”; When I asked him to support me to test an idea in fruit fly, “You should do it. It is
your life”. I told him that I practiced every week with the university choir where I perform
tenor, and then I found him and his family in the audience during the final concert. I
always ask myself: if one day I became a supervisor, can I be as supportive as Neil? I
think I will, at least I know who to learn from.
I also want to thank my long-term collaborator and hiking partner Juan Llamas, who
helps me accelerate my process of being “Americanized”. I appreciate the love and care
that Xing and I have received from his family, particularly from Juan’s parents, sisters
and Dawn. I hope we resume our annual Mt. Whitney hike sometime soon. In addition, I
want to thank everybody from Segil lab for their support, especially Welly Makmura,
Suhasni Gopalakrishman, and Litao Tao.
iv
I appreciate the opportunity to work with, and learn from Dr. Andy McMahon, a
world-class scientist; and Dr. Gage Crump, Dr. Francesca Mariani, Dr. Andy Groves,
three really brilliant developmental biologists. They have given me so many
opportunities to participate in their projects, and collaborate with excellent researchers
in their lab.
Finally I want to thank my parents, who help me to build up my confidence, and
support me both financially and emotionally. They are my greatest motivation to move
forward.
v
Table of Contents
Dedication ........................................................................................................................ ii
Acknowledgements .......................................................................................................... iii
List of Figures .................................................................................................................. vii
Abstract ........................................................................................................................... ix
Chapter 1 Introduction: The making of secondary receptor cells ..................................... 1
Chapter 2. A feed-forward ATOH1/POU4F3-dependent pioneer activity drives divergent
enhancer networks in inner ear hair cells and Merkel cells during mechanosensory cell
development ................................................................................................................... 18
Introduction ............................................................................................................... 18
Results ...................................................................................................................... 22
ATOH1 binds to “pre-established” and “de novo” distal regulatory elements in
differentiating hair cells ........................................................................................ 22
POU4F3 is necessary to provide ATOH1 access to its targetome in nascent hair
cells ..................................................................................................................... 26
POU4F3 binds closed chromatin in a heterologous cell type, and can synergize
with ATOH1 to stimulate open chromatin formation ............................................ 31
ATOH1 and POU4F3 synergize to form a feed-forward regulatory circuit in
differentiating hair cells ........................................................................................ 32
The ATOH1-POU4F3 feed-forward circuit is conserved in Merkel cells ............. 37
The ATOH1-POU4F3 synergy is conserved between mechanosensory cell types
with divergent enhancer networks ....................................................................... 39
Discussion ................................................................................................................ 44
Chapter 3: Enhancer decommissioning during inner ear maturation imposes an
epigenetic barrier to sensory hair cell regeneration ....................................................... 53
Introduction ............................................................................................................... 53
Results ...................................................................................................................... 55
Active enhancers around the hair cell genes are mostly ATOH1 targets ............ 55
Hair cell gene promoters are poised in the perinatal supporting cells ................. 58
vi
Hair cell-specific enhancers are also poised in the perinatal supporting cells .... 59
Low expression of hair cell-specific genes in supporting cells is maintained by
active histone de-acetylation and histone methylation ........................................ 61
Separating responding and non-responding supporting cells from perinatal
cochlea during transdifferentiation ...................................................................... 62
Supporting cell transdifferentiation is accompanied by activation of the poised
hair cell-specific enhancers, and the acetylation of the poised promoters .......... 63
Hair cell-specific gene promoters are kept bivalent during supporting cell
maturation ........................................................................................................... 65
Decommission of the hair cell-specific enhancer correlates with loss of
transdifferentiation potential in supporting cells .................................................. 66
Discussion ................................................................................................................ 71
Chapter 4: Conclusion .................................................................................................... 75
Chapter 5: Experimental procedures ............................................................................. 78
References ..................................................................................................................... 86
Appendix ...................................................................................................................... 102

vii
List of Figures
Figure 1. ATOH1 binds to pre-established and de novo distal regulatory elements in
differentiating hair cells .................................................................................................. 23
Figure 2. Correlation analysis at the promoters and distal elements detected in the
sensory progenitors and hair cells ................................................................................. 25
Figure 3. The de novo hair cell-enriched distal elements are not marked by any tested
histone modifications ...................................................................................................... 27
Figure 4. POU4F3 is necessary to provide ATOH1 access to its complete targetome .. 28
Figure 5. POU4F3 binds closed chromatin and facilitates ATOH1 binding at many
ATOH1-targets in a heterologous cell type .................................................................... 30
Figure 6. ATOH1 and POU4F3 synergize to form a feed-forward regulatory circuit in
differentiating hair cells .................................................................................................. 34
Figure 7. The ATOH1-POU4F3 feed-forward circuit is conserved in mechanosensory
Merkel cells .................................................................................................................... 36
Figure 8. The ATOH1-POU4F3 synergy represents a conserved gene regulatory
mechanism for differentiation of mechanosensory cells with divergent enhancer
networks ......................................................................................................................... 40
Figure 9. Gene ontology analysis of the POU4F3-dependent ATOH1 targets in hair cells
and Merkel cells ............................................................................................................. 41
Figure 10. Six1 is broadly expressed in the cochlea ...................................................... 43
viii
Figure 11. Analysis of the hair cell epigenome defines a class of “active” putative hair
cell enhancers at postnatal day 1, as well as the ATOH1 hair cell targetome ............... 56
Figure 12. The hair cell gene regulatory network is “primed”, but silenced by both
H3K27me3 and active HDAC activity in P1 supporting cells ......................................... 60
Figure 13. Transdifferentiation of supporting cells is accompanied by HC-gene enhancer
activation (H3K27ac) and increased chromatin accessibility (ATACseq) ...................... 64
Figure 14. Decommissioning of HC-gene enhancers (H3K4me1) accompanies postnatal
maturation of supporting cells ........................................................................................ 77
Figure 15. The “primed” state of hair cell-gene enhancers (H3K4me1) in supporting cells
determines the transcriptional response during transdifferentiation (Notch inhibition) ... 70

ix
Abstract
Neurosensory cells are epithelial cells with neuronal characteristics including being
excitable by both internal and external stimuli. In vertebrates, neurosensory cells such
as hair cells, gustatory cells, and Merkel cells are specialized for the senses of hearing,
balance, taste and light touch. Aside from these, there are many types of
neuroendocrine cells in the internal organs. For example, pulmonary neuroendocrine
cells (PNECs) in the airway epithelium and enteroendocrine cells in the gut collaborate
with immune cells and neural circuits, in order to respond to signals in the inhaled air or
ingested contents. Despite their different embryonic origins and their division of labor in
perceiving stimulants, these neurosensory cells share a striking array of similarities at
the cellular and molecular level, including the expression of ion channels,
neurotransmitters, synaptic proteins, cilium components, etc.
Hearing and balance are mediated in vertebrates by inner ear mechanosensory hair
cells. Hair cell development, maturation, and survival require the expression of ATOH1,
a bHLH transcription factor that is considered a master regulator of hair cell
differentiation. However, we now show that at the time of hair cell differentiation, a large
percentage of the ATOH1 targetome lies in “closed” chromatin, to which ATOH1 is
unable to bind. We show that one of the first direct targets of ATOH1 expressed in hair
cells is POU4F3, a class IV POU-domain transcription factor that is also necessary for
hair cell differentiation. ATOH1 can bind to several open enhancers at the Pou4f3 locus,
and activates its early expression in hair cell precursors. Unlike ATOH1, we show that
POU4F3 can bind “closed”, nucleosomal DNA, and has pioneer factor activity needed to
x
remodel the closed ATOH1 targetome, allow access to ATOH1, and thereby promotes
hair cell differentiation in a feed-forward manner. We demonstrate that this feed-forward
mechanism is also necessary for the differentiation of Merkel cells, a mechanosensory
cell population in the skin responsible for mediating light touch. Although the
transcriptomes of hair cells and Merkel cells are very different, they share many
POU4F3 and ATOH1 chromatin targets, suggesting that different vertebrate
mechanoreceptors may use elements of the same ancient epistatic gene regulatory
mechanism, supplemented by distinct enhancer networks, to produce specific
mechanoreceptive cell types.
One of the striking differences between the hair cells and the other secondary
receptor cell types in mammals is regenerative capability. No adult stem cells are
present in the mammalian inner ear, and death of mechanosensory hair cells fails to
elicit regeneration from the surrounding supporting cells. In contrast, supporting cells in
non-mammalian vertebrates are able to divide and transdifferentiate into hair cells to
restore function following hair cell loss. A latent potential for supporting cell direct
transdifferentiation can be elicited in the neonatal organ of Corti of mice by blocking
Notch-mediated lateral inhibition, suggesting that the gene regulatory network governing
transdifferentiation remains intact in perinatal supporting cells. By profiling chromatin
accessibility and histone modifications in mouse hair cells and supporting cells, we
show that hair cell gene enhancers primed by H3K4me1 are silenced in supporting cells
by H3K27 trimethylation and active deactylation. These marks are rapidly reversed
during neonatal supporting cell transdifferentiation in response to Notch inhibition.
xi
However, this latent transdifferentiation potential is lost by the end of first postnatal
week. We report that loss of transdifferentiation potential is accompanied by a loss of
H3K4me1-priming at hair cell gene enhancers in supporting cells. We further show that
blocking H3K4me1 removal leads to a temporal extension of transdifferentiation
potential. We hypothesize that this enhancer decommissioning contributes to the failure
of supporting cell regeneration in the mature organ of Corti.
1
Chapter 1. The making of secondary receptor cells
The increasing sensitivity and resolution of the sensory organs during evolution is
accompanied by the acquisition of the axon-less secondary receptor cells
(neurosensory cells), as the primary sensory transduction sites, coupled with afferent
neurons projecting to their central targets. Whereas the primordial sensory modules,
commonly found among the invertebrates, comprise only the primary sensory neurons
and the associated supporting cells.
In vertebrates, there is a large diversity of neurosensory cells, and they specialize in
detecting various external and internal stimuli, record different development origins, and
reside at distinct locations. Some are found in specialized sensory organs. For example,
hair cells in the inner ear are responsible for the senses of hearing and balance, and
originate from placodal ectoderm (Groves et al., 2013); taste cells on the tongue
epithelium are responsible for the sense of gustation, and originate from both ectoderm
and endoderm (Okubo et al., 2009); Merkel cells in the epidermis are responsible for
sense of light touch, and originate from non-neural ectoderm (Maricich et al., 2009;
Morrison et al., 2009; Van Keymeulen et al., 2009), etc. Interestingly, many types of
neurosensory cells are identified in the internal organs, which render the sensory
functions of these organs. For example, pulmonary neuroendocrine cells (PNECs) in the
airway epithelium and enteroendocrine cells in the intestine collaborate with immune
cells and neural circuits, in order to respond to signals in the inhaled air or ingested
contents (Garg et al., 2019). In addition, glomus cells in carotid bodies are hypoxia-
2
sensors to monitor the oxygen level in the carotid artery, and originate from neural crest
(Hockman et al., 2017).
Despite their different locations, embryonic origins and division of labor in perceiving
various stimulants, these neurosensory cells share a striking array of similarities at the
cellular and molecular level, including the expression of ion channels, neurotransmitters,
synaptic proteins, cilium components and other epithelium characteristics, etc. For
example, PNECs and enteroendocrine cells, which are originally thought to act
exclusively remotely through secreting hormones, are recently found to be directly
innervated by sensory neurons (Bellono et al., 2017; Kaelberer et al., 2018). In addition,
many epithelial receptors for canonical senses also possess endocrine features. Merkel
cells, the epithelial sensors for gentle touch, express both neuropeptide CGRP, and
neurotransmitters such as glutamate and norepinephrine (Higashikawa et al., 2019;
Hoffman et al., 2018; Lucarz and Brand, 2007). These observations suggest that the
dual functions of these neurosensory epithelial cells to produce, store and release both
slow-acting local hormones and fast-acting neurotransmitters may be controlled by a
common gene regulatory circuit. At the molecular level, the generation of these
neurosensory cells unanimously involves the inputs from various basic helix-loop-helix
(bHLH) transcription factors. For example, the specification of the placodal hair cells,
the ectodermal Merkel cells, and the endodermal enteroendocrine cells require the
expression of Atoh1, while Ascl1 controls the differentiation of endoderm-derived
PNECs and neural crest-derived glomus cells. There observations suggest that the
3
phenotypic convergence of the neurosensory cell types may reflect the shared
epigenomic grammar for gene expression associated with their common features.
The making of taste cells
The sense of gustation in mammals is mediated by taste cells located in the oral and
pharyngeal cavities. The majority of the taste cells are grouped in the taste buds as 60-
100 cell aggregates, resided in the lingual epithelium, and associated with specialized
epithelial appendages called taste papillae (Barlow, 2015; Barlow and Klein, 2015; Feng
et al., 2014; Kapsimali and Barlow, 2013). While the other taste cells can be found in
the soft pallet and larynx. There are three types of taste papillae on the dorsal surface of
the tongue (Witt and Reutter, 2015). Fungiform papillae are scattered in the anterior 2/3
of the lingual epithelium. Each fungiform papilla contains only one taste bud. Posteriorly,
a single central circumvallate papilla and numerous bilateral foliate papillae house
hundreds of taste buds, located along the invaginated epithelial trenches, surrounding
the bulged mesenchymal papilla core.
Regardless of their location in the tongue, in each taste bud, there are at least five
physiologically defined taste cells, which are reactive correspondingly to five basic taste
stimuli: salt, sour, sweet, bitter and umami (Chandrashekar et al., 2010; Liman et al.,
2014). While conventionally taste cells are classified morphologically into three
subtypes (Finger and Simon, 2000). Type I taste cells are glial-like supporting cells,
which are the most abundant subtype. These cells do not express taste receptors, but
closely wrap around the other subtypes with their extensive cellular processes. The
functions of Type I taste cells are poorly studied. Type II taste cells are responsible for
4
the senses of sweet, bitter and umami, with each of the taste stimulus detected by
individual subtypes of Type II cells, expressing different combinations of taste receptor
genes. Type III taste cells are the only taste cell type that harbors the presynaptic
structures, thus is directly innervated by sensory neurons (Chandrashekar et al., 2010;
Yang et al., 2004). These cells transduce sour and salty stimuli, and are the least
abundant taste cell types in the buds. Lastly, Type IV taste cells (basal cells) are located
at the base of the taste buds. In adults, these cells serve as the postmitotic progenitors
for all the other cell types in the buds (see below).
During embryonic development, cells in the taste buds are derivative of the local
epithelia originated from both ectoderm and endoderm, but not the neural crest (Stone
et al., 1995). The taste cells in the anterior fungiform papillae are ectoderm-derived,
while those associated with the more posterior circumvallate and foliate papillae are
endoderm-derived (Rothova et al., 2012). In spite of their embryonic origins, the
specification of taste cells in the buds during development follows the process of taste
placode induction from the local epithelium, which is evident by their columnar
morphology standing out of the rest of the cuboidal lingual epithelial cells (Barlow, 2015;
Kapsimali and Barlow, 2013). At around embryonic day 13.5 (E13.5) in mouse, Sonic
hedgehog (Shh) is among the first identified marker genes specifically expressed in the
taste placodes but not the surrounding epithelial cells (Thirumangalathu et al., 2009).
These Shh-expressing placodal progenitors differentiate exclusively into taste cells that
form the embryonic taste buds, but do not contribute to the taste papillae that surround
the taste buds.
5
During postnatal maturation and in the adults, as the placodally-derived cells are lost
in the taste buds, taste cells are continuously replenished by new cells entering the
taste buds from basal region outside the buds, where the basal progenitors are marked
by the co-expression of Krt14, Krt5, Trp63 and Sox2 (Okubo et al., 2009;
Thirumangalathu et al., 2009). Once entering the taste buds, these new cells rapidly
turned off the expression of Krt14, Krt5 and Trp63, and subsequently become the Type
IV taste cells, the basal cells in the buds (Thirumangalathu et al., 2009). Interestingly,
these taste bud basal cells also express shh, the marker gene for the cells in the taste
placode during embryonic development, suggesting that these shh-expressing basal
cells may function as the precursors for the rest of the taste cell subtypes in adults.
Consistent with this idea, lineage-tracing experiments suggest that Shh-expressing
basal cells can give rise to at least 2 types (Type I and Type II) of taste cells. The failure
of detecting Type III cells as the basal cell-descendants might be because Type III taste
cells are long-lived cell type whose renewal is less likely to be detected during the
lineage-tracing experiments (Hamamichi et al., 2006; Perea-Martinez et al., 2013).
Compare to the current understandings of the signal transduction in taste cells in
response to gustatory stimuli (Liman et al., 2014; Roper and Chaudhari, 2017), the gene
regulatory networks that control the specification and differentiation of the three
functional taste cell subtypes from the taste bud basal cells are poorly understood.
Limited evidence suggests that transcription factor SOX2 is important for the proper
formation of taste papilla and taste bud (Castillo-Azofeifa et al., 2018; Okubo et al.,
2006), while ASCL1 and POU2F3 are crucial cell fate determinants for individual taste
6
cell types (Kito-Shingaki et al., 2014; Maeda et al., 2017; Matsumoto et al., 2011; Seta
et al., 2011). Other transcription factor genes, e.g. Ascl2, NeuroD1, Nkx2.2, Hes6, etc.,
are also expressed in the taste buds (Ren et al., 2017; Suzuki et al., 2002). However,
their functions are not known.
During embryonic development, Sox2 is expressed in both taste placode and, to a
lesser extent, the surrounding epithelia (Okubo et al., 2006). In a Sox2 hypomorphic
mouse model, fungiform papillae were normally formed initially at around E14.5, but
their number gradually decreased, and was completely absent at birth (Okubo et al.,
2006). These data demonstrated that high SOX2 protein level is required for both the
maintenance of taste papillae, and the formation of taste bud cells during development.
As mentioned above in the adult mice, Sox2 is also mildly expressed in the basal
progenitors of the non-taste epithelium adjacent to the taste buds that contribute to the
renewal of both the keratinocytes and taste cells (Ohmoto et al., 2017; Okubo et al.,
2009). The expression of Sox2 is upregulated in both immature (Type IV) and some
mature taste cells (Okubo et al., 2006). Therefore, it is likely that Sox2 is required for
both the maintenance of the progenitor pool, and the differentiation of taste cells during
homeostasis. Consistent with this hypothesis, conditionally knocking out Sox2 in K14-
expressing basal cells in adults disrupted the renewal of both the taste buds and the
non-taste keratinocytes (Castillo-Azofeifa et al., 2018).
Although lineage-tracing experiments showed that Shh-expressing basal cells failed
to give rise to Type III taste cells (Thirumangalathu et al., 2009), recent studies suggest
a common lineage between the Type II sweet/bitter/umami taste cells and Type III
7
sour/salty taste cells (Matsumoto et al., 2011). Knocking out Pou2f3 expression
completely abolished the specification of all three subtypes of Type II cells in the taste
buds, resulting in the expansion of Pkd2l1-expressing Type III cells, but not the other
taste cell types (Maeda et al., 2017; Matsumoto et al., 2011). Consistently, mutating
Ascl1a in zebrafish prevented the formation of serotonin-expressing Type III-like taste
cells, and resulted in the expansion of Calb2b-expressing Type II-like taste cells
(Kapsimali et al., 2011). However, it remains unclear whether Ascl1 is the cell fate
determinants of all Type III taste cells in the mammalian taste buds, or only a subtype of
Type III taste cells (Kito-Shingaki et al., 2014; Seta et al., 2011). Interestingly, both
Pou2f3 and Ascl1 were found to be upregulated in subgroups of basal cells in the taste
bud (Matsumoto et al., 2011; Seta et al., 2011), suggesting these transcription factors
are likely to be among the first upregulated genes during the specification of individual
subtype of taste cells.
The making of Merkel cells
The skin is our largest sensory organ that harbors a broad range of low threshold
mechanoreceptors that relay the sense of touch (Owens and Lumpkin, 2014;
Zimmerman et al., 2014), which require their expression of Piezo2, a rapidly adapting,
mechanically activated cation channel (Coste et al., 2010, 2012; Ranade et al., 2014).
While the Meissner corpuscles in the hairless skin are specialized for the detection of
the gentlest forces and the fine sensorimotor control (Neubarth et al., 2020), Merkel cell-
neurite complexes in both hairy and hairless skin are indispensable for shape, texture
and curvature discrimination, in addition to two points differentiation (Maricich et al.,
8
2012). Merkel cell-neurite complex is an excellent example of a two-receptor-one-site
model, in which both the mechanosensitive Merkel cells and the afferent terminals are
required for the proper slowly adapting type I (SA-I) firing patterns that encodes tactile
discrimination (Ikeda et al., 2014; Maksimovic et al., 2014; Woo et al., 2014a, 2015). In
Merkel cells, Piezo2 and uncharacterized voltage-gated ion channels (Maksimovic et
al., 2014), once activated, stimulate the release of neurotransmitters (Hoffman et al.,
2018) that tune mechanosensitive afferents to maintain both the static phase firing
throughout the stimulation, and the high-frequency firing that are important for detecting
shape, texture and curvature, etc. (Maksimovic et al., 2014; Woo et al., 2014a). Without
Merkel cells, touch dome afferents lose the SA-I pattern, resulting in moderately
decreased behavioral responses to gentle touch (Maricich et al., 2009a, 2012; Woo et
al., 2014a).
During embryonic development, the induction of Merkel cell fate in the hairy skin is
closely associated with the formation of the hair pegs of the guard hair follicles (Nguyen
et al., 2018; Vielkind et al., 1995; Xiao et al., 2016). At around E13.5 in mice, Merkel
cells first appear and scatter within the developing hair follicles, expressing their specific
marker genes Atoh1, Sox2 and Krt8. Subsequently at around E15.5, as more Merkel
cells are specified outside but adjacent to the elongating primary hair placodes (Xiao et
al., 2016), Merkel cells begin to migrate and form crescent-shaped aggregates between
the epidermis and the dermis, caudal to the primary hair follicles (Vielkind et al., 1995),
where the rudimental touch domes form. These observations suggest that Merkel cells,
unlike other sensory epithelial receptors such as hair cells and taste cells, do not derive
9
from sensory placodes, but are directly induced from the local epithelium (Jenkins and
Lumpkin, 2017). Indeed, studies showed that although hair follicle-produced Shh is
required for Merkel cell induction during embryonic stages, Shh agonist is sufficient to
rescue Merkel cell differentiation in the skin deficient for hair placode formation
(Perdigoto et al., 2014; Xiao et al., 2016). In addition, lineage-tracing experiments
confirmed that Merkel cells are derived from non-neural ectoderm, but not the other
lineages including the neural crest (Morrison et al., 2009; Van Keymeulen et al., 2009).
Interestingly, the homeostasis of Merkel cells and their progenitors in the touch
domes in adults (Doucet et al., 2013; Woo et al., 2010) are also dependent on Shh, but
from a different source (Xiao et al., 2015). Adult Merkel cells have a life span of about 5
months (Wright et al., 2017), and are replenished by a group of bipotent progenitors that
reside within the touch domes, which also give rise to the touch dome keratinocytes.
These touch dome progenitors are phenotypically distinct from the basal cells of the
surrounding epidermis (Doucet et al., 2013; Woo et al., 2010; Xiao et al., 2015),
including their expression of Krt17 and their activated Shh signaling. Deleting Shh
expression in the dorsal root ganglion (DRG) neurons that innervate the touch dome did
not affect the numbers and the distribution of Merkel cells in the touch dome at birth.
However, lack of DRG neuron-derived Shh resulted in the continuous reduction in the
size of the touch domes and the numbers of Merkel cells during postnatal development
(Xiao et al., 2015). These data suggest that the DRG neurons, not the hair follicles are
the primary source of Shh for touch dome homeostasis in the adults.
10
In addition to their function as the adult stem cells, touch dome epithelial progenitors
are also required for maintaining the proper innervation of Merkel cells. Genetic ablation
of Krt17-expressing cells, not the ablation of the presynaptic Merkel cells, resulted in
severe deterioration of afferent fibers in the touch domes (Doucet et al., 2013). This
observation is consistent with the finding that Merkel cells are not required for neither
the formation of touch domes, nor their innervation during embryonic development
(Maricich et al., 2009a). These data fit the hypothesis that Merkel cells may have
emerged through the incorporation of a conserved gene regulatory mechanism for
mechanosensation into the ectoderm-derived non-sensory epithelial cells during
evolution (Arendt et al., 2016).
Atoh1 and Sox2 are among the first expressed transcription factors during Merkel
cell specification (Lesko et al., 2013; Maricich et al., 2009a; Perdigoto et al., 2014; Van
Keymeulen et al., 2009). Unlike in hair cells and taste cells, where Sox2 is expressed in
the sensory placodes before the specification of the sensory receptor cells, Sox2
expression in Merkel cells seems to come after (downstream of) Atoh1 expression
(Perdigoto et al., 2014). Conditional knocking out Atoh1 in the epidermis prevented the
expression of all known Merkel cell marker genes, including Sox2, while Atoh1 and Krt8
were still expressed in the Sox2 mutant Merkel cells. However, the number of Merkel
cells in Sox2 mutant mice decreased dramatically compared to those in the controls
(Perdigoto et al., 2014). Since immature Merkel cells are mitotically active (Wright et al.,
2015), these data suggest that Sox2 controls the proliferation of immature Merkel cells
downstream of Atoh1. In addition, Krt20, a mature Merkel cell keratin gene, and Rab3c,
11
a synaptic vesicle component gene (Haeberle et al., 2004) were not detected in Sox2
mutant Merkel cells (Bardot et al., 2013; Perdigoto et al., 2014), indicating that Sox2
also regulates Merkel cell maturation and functional differentiation. Many other
transcription factors are also known to be expressed in the Merkel cells, but their
functions remain uncharacterized (Haeberle et al., 2004).
The making of PNECs
Pulmonary neuroendocrine cells (PNECs) are rare cell types (< 0.5%) present in the
conductive airway epithelium (the trachea and the bronchus) that potentiate the
mammalian lung as a sensory organ (Garg et al., 2019). The functions of PNECs have
been associated with the detection of varying O2/CO2 levels and certain chemical
signals enriched in aerosols, including pollutants, allergens, etc., as well as physical
stimuli such as cyclic stretching (Lembrechts et al., 2012; Pan et al., 2006). PNECs are
thought to respond to these stimulants by collaborating with immune cells and neural
circuits (Garg et al., 2019). While most of these sensory functions of PNECs were
implicated from results of the in vitro experiments (Lauweryns et al., 1977; Schüller et
al., 1990), recent in vivo studies demonstrated that PNECs are required for amplifying
type II immune response in asthmatic models in mice (Sui et al., 2018). Knocking out
PNECs in the airway epithelium weakened immune cell infiltration, at the same time
dampened goblet hyperplasia upon allergen challenge. Consistent with these findings,
excessive numbers of PNECs were found in the lungs of human asthma patients,
suggesting increased PNEC products, such as the neuropeptide CGRP and the
inhibitory neurotransmitter GABA, may contribute to allergic asthma (Sui et al., 2018). In
12
addition, PNECs also produce serotonin and ATP (Cutz et al., 1993), and form direct
synaptic connections with vagal and sensory neurons (Chang et al., 2015; Gu et al.,
2014; Pan et al., 2004). However, it remains unclear what behavioral and physiological
aspects are dependent on the synaptic transmission and sub-second signaling from
PNECs (Hoffman and Lumpkin, 2018).
In humans, PNECs are found mostly as solitary chemosensory cells scattered in the
pseudostratified airway epithelium (Weichselbaum et al., 2005). In mice, the distribution
of PNECs in the trachea is similar to those in the human lung. However, in the murine
bronchi, PNECs form clusters of ~5-20 cells called neural epithelial body (NEB), located
primarily at the branch points, ideal locations for aerosol signal detection where the
inhaled particles congregate (Kuo and Krasnow, 2015; Noguchi et al., 2015; Sui et al.,
2018). At around E15 in mice, PNECs are initially specified randomly throughout the
bronchial epithelium, but rapidly form clusters after transient epithelial-to-mesenchymal-
transition (EMT) (Kuo and Krasnow, 2015; Noguchi et al., 2015). This targeted migration
of PNECs to the branch points, termed “slithering” (Kuo and Krasnow, 2015), is
dependent on Slit-Robo signaling pathway (Branchfield et al., 2016). Interestingly,
formation of PNEC clusters during early development also regulates the proper level of
immune response in the lung upon air exposure. Conditionally mutating Roundabout
receptor (Robo) genes or their ligand gene slit in the airway epithelium or PNECs not
only prevented the formation of PNEC organoids in vivo, but also triggered excess
neuropeptides production from the failed-to-cluster PNECs, leading to increased
13
immune filtration that irreversibly simplified the alveoli during postnatal development in
mice (Branchfield et al., 2016).
During embryonic development, PNEC is among the first emerged differentiating cell
types from the embryonic airway epithelium (Branchfield et al., 2016). Lineage tracing
experiments confirmed at PNECs are derived from endoderm-originated Shh-
expressing local epithelium (Kuo and Krasnow, 2015; Noguchi et al., 2015; Song et al.,
2012; Sui et al., 2018), instead of once believed neural crest descendants (Hockman et
al., 2017). In the adult human airway and in the adult mouse trachea, PNECs are long-
lived and are slowly replenished by Krt14/Krt5-expressing basal cells (Montoro et al.,
2018; Plasschaert et al., 2018). In contrast, in the murine bronchial epithelium where
basal cells are lacking, PNECs, once genetically ablated, cannot be generated from
other epithelial cell types (Song et al., 2012), suggesting PNEC self-renew occurs within
the NEB. Strikingly, recent studies showed that a subgroup of PNECs in the NEB have
progenitor properties, which can differentiate into club cells and ciliated cells after
Naphthalane-induced airway epithelial damage (Ouadah et al., 2019; Song et al., 2012).
Recent single cell analysis of the adult airway epithelium in both human and mouse
revealed many transcription factors expressed in PNECs, including Ascl1, Gfi1, Sox1,
Foxa2, etc. (Montoro et al., 2018; Plasschaert et al., 2018). However, the gene
regulatory network that governs the differentiation and maturation of PNECs remains
uncharacterized. The specification of PNECs from the embryonic airway epithelium
requires the expression of the bHLH transcription factor Ascl1 (Borges et al., 1997; Ito
et al., 2000), the same gene that is proposed to be the master regulator for Type III
14
taste cell differentiation. Although it is not clear whether Ascl1 is required for the
maintenance of PNEC cell fate in adults. Another transcription factor, Gfi1, is specifically
expressed in PNECs in the airway epithelium, as well as other neurosensory cell types
including hair cells, Merkel cells, taste cells, in addition to the majority of the
neuroendocrine tumors (Kazanjian et al., 2004; Wallis et al., 2003). The specification of
PNECs and their Ascl1 expression level was not affected in Gfi1-mutant airway
epithelium, suggesting Gfi1 is likely a downstream transcription factor of Ascl1 in
PNECs. In contrast, the expression of mature PNEC markers, including CGRP and SYN,
were dramatically decreased without Gfi1 expression (Kazanjian et al., 2004; Linnoila et
al., 2007), suggesting Gfi1 is a crucial transcription factor for PNEC maturation.
The making of hair cells
Hair cells in the inner ear are specialized for the detection of fluid movement.
Cochlear hair cells are responsible for the sense of hearing, while the hair cells found in
the vestibular sensory epithelia mediate different aspect of the sense of balance. Both
cochlear and vestibular hair cells originate from a thickened ectodermal tissue around
the hindbrain regions called the otic placode, which then invaginate to form otic vesicle
(Groves et al., 2013). The dorsal portion of the otic vesicle develops to form the
vestibule, while the ventral otic vesicle expands to form the cochlea. At the same time,
some cells delaminate from the otic vesicle, and differentiate into the precursors of the
VIIIth cranial nerve, which subsequently innervate the cochlear and vestibular hair cells,
and relay the sense of hearing and balance to the brain.
15
Like Merkel cells, the specification of hair cells in the inner ear also relies on the
expression of Atoh1. At around E12.5, as the cochlear duct elongates, Cdkn1b (P27
Kip1
),
a gene that encodes a cell cycle inhibitor, start to express at the apex of the cochlea,
and propagate to reach the basal cochlea at around E13.5. The wave of cell cycle exit
follows the expression of Cdkn1b, therefore marking the domain of postmitotic cells,
which contains the sensory progenitors that will later on differentiate as both cochlear
hair cells and supporting cells (Chen and Segil, 1999; Lee et al., 2006). At around E13.5
to E14.5, Atoh1 is upregulated in a group of progenitor cells at the base of the cochlea,
and the expression of Atoh1 propagate in a basal-to-apical gradient, which is opposite
to the gradient of the formation of prosensory domain (Chen et al., 2002; Maricich et al.,
2009a). Atoh1 expression is required for both the differentiation and survival of the cells
in the prosensory domain. The mechanisms that result in the upregulation of Atoh1 at
the base of the cochlea remain unclear. Another intriguing question is how is the
specification of the postmitotic prosensory domain regulated to follow an apical-to-basal
gradient.
SOX2 is one of the first identified transcription factors necessary for the sensory
differentiation in the inner ear (Kiernan et al., 2005a). Using a Sox2
CreER
/ Sox2
flox

mouse line, in which tamoxifen injection leads to the deletion of Sox2 gene in the Sox2
expressing cells (Steevens et al., 2019), it was found that deletion of Sox2 at around
E12.5 completely abolished the differentiation of hair cells in the inner ear, while the
morphologies of the cochlea and the vestibular organs are perfectly normal. The failure
of hair cell formation in the cochlea in Sox2 conditional mutant is likely due to the failure
16
of prosensory domain formation, since the Cdkn1b failed to upregulate without Sox2
expression. In addition, overexpression of Sox2 in the non-sensory regions of the
cochlea can induce Atoh1 expression, suggesting that SOX2 also plays important roles
during hair cell differentiation (Dabdoub et al., 2008). Interestingly, the epistatic
relationship between Sox2 and Atoh1 are completely different in hair cells and Merkel
cells, since the specification of Atoh1+ Merkel cells is not dependent on presence of
SOX2 (Perdigoto et al., 2014).
One of the striking differences between hair cells and the other secondary receptor
cell types in mammals is the formation of the stereocilia, several connected rows of
elongated microvilli with graded length, present in a polarized manner at the apical
surface of the hair cells. In mice, there are three rows of stereocilia, and in humans
there are four rows. Specialized transmembrane proteins form spring-like structures,
called tip links, connecting the tip of the shorter stereocilium to the sidewall of the longer
stereocilium. The tip-links are also coupled with the proteins that form the
mechanotransduction channel complexes at the tip of the shorter stereocilium.
Therefore, deformation of the longer stereocilium will lead to the tension of the tip-links,
which will force the opening of the mechanotransduction channels to allow ions to flow
into the hair cells. As a result, hair cells depolarize and release neurotransmitters
(Pepermans and Petit, 2015; Petit and Richardson, 2009).
The gene regulatory networks that control the expression of cilia components and
the mechanotransduction channel complexes are of great interests. Since ATOH1 is the
master regulator for hair cell differentiation, it was believed that Atoh1 also controls the
17
ciliagenesis of the hair cell by regulating the expression of the associated genes (Cai et
al., 2015). However, Atoh1 is only transiently expressed in the cochlear hair cells, and it
is also transiently expressed in one of the two types of vestibular hair cells (Type I hair
cells). Although it is not clear what mechanism controls the downregulation of Atoh1 in
the cochlear hair cells and type I vestibular hair cells, it was found that the
downregulation of Sox2 in these hair cell subtypes correlates with their downregulation
of Atoh1. Other transcription factors, including Pou4f3, Lhx3, Barhl1, Gfi1, Rfx3, were
also shown to regulate the proper formation of the stereocilia (Elkon et al., 2015;
Hertzano et al., 2004; Wallis et al., 2003; Xiang et al., 1997a, 1998; Zhong et al., 2018).
However, their exact mechanisms at the molecular level remain uncharacterized.
18
Chapter 2. A feed-forward ATOH1/POU4F3-dependent
pioneer activity drives divergent enhancer networks in inner
ear hair cells and Merkel cells during mechanosensory cell
development
INTRODUCTION
Mechanosensory hair cells of the inner ear are secondary receptor cells that deliver
sound and balance stimuli to the brain through synaptic connections with primary
sensory neurons. Hair cells and the supporting cells that surround them, develop from
distinct regions of auditory and vestibular progenitors in the embryonic inner ear
(Atkinson et al., 2015; Groves et al., 2013; Kelley, 2006). The first sign of hair cell
differentiation in these prosensory regions is the expression of ATOH1, a bHLH
transcription factor that is necessary and sufficient for hair cell differentiation
(Bermingham et al., 1999; Cai et al., 2015; Chen et al., 2002; Woods et al., 2004; Zheng
and Gao, 2000). Subsequently, Atoh1 expression is inhibited in adjacent sensory
progenitors and later in immature supporting cells by Notch-mediated lateral inhibition
(Abdolazimi et al., 2016; Kiernan et al., 2005b; Lanford et al., 1999; Tateya et al., 2011),
which patterns the auditory and vestibular sensory epithelia into stereotyped mosaics of
hair cells and supporting cells (Figure 1A).
Several independent lines of evidence suggest that most sensory progenitors in the
developing cochlea are competent to express and respond to ATOH1. First, in utero
electroporation of Atoh1 into the embryonic inner ear significantly increases the number
of hair cells at the expense of supporting cells (Gubbels et al., 2008). Second, genetic
19
or pharmacological disruption of Notch-mediated lateral inhibition in the embryonic
cochlea causes an increase in Atoh1-expressing hair cells, again at the expense of
supporting cells (Doetzlhofer et al., 2009; Kiernan et al., 2005b). Third, lineage tracing
with Atoh1-Cre and Atoh1-CreER knock-in mice suggest that many sensory progenitor
cells that initially express Atoh1 ultimately differentiate as supporting cells (Driver et al.,
2013; Yang et al., 2010). One possible explanation for this broad sensory competence
is that chromatin at distal regulatory elements of hair cell gene loci, including direct
targets of ATOH1, is already accessible in sensory progenitors and may be primed by
post-translational histone modifications associated with poised and active enhancers,
such as H3K4me1 (Calo and Wysocka, 2013; Kim et al., 2014). However, it has hitherto
been difficult to analyze these epigenetic states and identify elements bound by ATOH1
during differentiation of cochlear sensory progenitors due to their scarcity, since
embryonic cochlea contains only a few thousand progenitor cells.
Several other transcription factors, including a class IV POU domain transcription
factor, POU4F3, are co-expressed during the first stages of hair cell differentiation
(Xiang et al., 1997b, 1998). Pou4f3 is thought to be a direct target of ATOH1 in
developing hair cells (Cai et al., 2015; Ikeda et al., 2015), and is necessary for their
differentiation, maturation and survival (Hertzano et al., 2004; Xiang et al., 1997b,
1998). Mutations of Pou4f3 cause human autosomal dominant, non-syndromic hearing
loss DFNA15 (Vahava et al., 1998). In addition to being necessary for hair cell
development, ectopic co-expression of Atoh1 and Pou4f3 is able to promote the
formation of a limited number of supernumerary hair cells in the mature cochlea
20
(Walters et al., 2017). However, the molecular and epigenetic mechanisms by which
ATOH1 and POU4F3 render hair cell gene loci accessible and transcriptionally active
are unknown.
In chapter 2, we used CUT&RUN (C&R, Skene and Henikoff, 2017) to identify the
distal regulatory elements of differentiating hair cells that bind ATOH1. We used
ATACseq (Buenrostro et al., 2013) to show that the majority of these ATOH1-bound
distal elements fall within nucleosome-occupied, silent chromatin in hair cell progenitors.
We show that POU4F3 binds nucleosomal DNA, and regulates the chromatin
accessibility at ~45% of “de novo” ATOH1 targets in differentiating hair cells. In Pou4f3
mutant hair cells, these POU4F3-dependent enhancer elements remain closed, and
ATOH1 is unable to access them. In addition, the expression levels of ATOH1 target
genes, including many deafness genes such as Grxcr1, Atp8a2, Adcy1, Ush2a, Myo3b,
are significantly reduced. These results suggest a feed-forward mechanism by which a
conventional transcription factor (ATOH1) can expand its targetome in silent chromatin
by directly activating the expression of a pioneer factor, POU4F3. Finally, we show this
ATOH1/POU4F3-dependent feed-forward mechanism is conserved in another
vertebrate mechanosensory cell type, Merkel cells, which mediate light touch in the skin
(Ikeda et al., 2014; Maksimovic et al., 2014; Woo et al., 2014b). We identify several
hundred POU4F3-dependent ATOH1 targets present in both hair cells and Merkel cells,
in addition to large numbers of cell-type-specific targets. Our results suggest that
POU4F3 and ATOH1 may be part of an ancestral core gene regulatory mechanism for
mechanosensory differentiation whose enhancer network subsequently expanded and
21
diversified to carry out the unique functions of these specialized epithelial cells.
22
RESULTS
ATOH1 binds to “pre-established” and “de novo” distal regulatory elements in
differentiating hair cells
We purified embryonic day 13.5 (E13.5) Cdkn1b+ (p27
Kip1
) sensory progenitors, and
E17.5 Atoh1+ nascent hair cells from the embryonic mouse cochlea using reporter mice
expressing GFP (Figure 1A) (Lee et al., 2006; Rose et al., 2009), and performed
transcriptomic and epigenetic analysis on each population. We adapted the ATACseq
technique (Buenrostro et al., 2013) to profile chromatin accessibility with small numbers
of FACS-purified cells from the embryonic cochlea (µATACseq, Appendices). We used
μATACseq to investigate the overall changes in chromatin structure during the transition
from Cdkn1b-GFP+ cochlear sensory progenitors at E13.5, to nascent ATOH1-GFP+
hair cells at E17.5. We identified 16,006 open promoters, and found that open promoter
regions were highly correlated between E13.5 sensory progenitors and E17.5 hair cells
(R=0.81, Figure 2A, B), suggesting that changes in promoter accessibility can’t on their
own explain the transition from E13.5 progenitors to E17.5 hair cells.
We next analyzed the changes in accessibility at distal elements to further explore
the transcriptional regulatory mechanisms that control the transition from E13.5
progenitors to E17.5 hair cells. We detected 64,803 distal elements from both progenitor
and hair cell populations and observed that in contrast to our promoter analysis, the
accessibility of distal elements was highly dynamic (R=0.41, Figure 1B and Figure 2C,
D). We found that 21,662 distal elements were less accessible in E17.5 hair cells
relative to E13.5 progenitors, while 18,681 distal elements were significantly more
23
Figure 1. ATOH1 binds to pre-established and de novo distal regulatory elements in differentiating hair cells. (A)
Schematic of early mouse organ of Corti development: Photomicrograph of E13.5 cochlear whole mount expressing
Cdkn1b-GFP within the prosensory domain represented by an equivalence group of undifferentiated sensory
progenitors. Through Atoh1-dependent, Notch-mediated lateral inhibition, this group differentiates into a mosaic of
hair cells and supporting cells, as seen in a photomicrograph of an E17.5 cross-section of organ of Corti expressing
ATOH1-GFP in hair cells, and stained with antibody to the hair cell marker MYO7A, and counter-stained by DAPI.
Scale bar: 50 μm. (B) Venn diagram illustrating the quantitative comparison of chromatin accessibility at the 64,803
common and unique distal elements in sensory progenitors and nascent hair cells (n=2; False discovery rate < 0.1).
18,681 distal elements are significantly more accessible in the nascent hair cells (hair cell-enriched distal elements)
relative to sensory progenitors (MACS2 for peak calling and DESeq2 for statistical analysis). (C) Hair cell-enriched
distal elements (18,681) are shown in heatmaps sub-clustered into “pre-established” and “de novo” sites based on
their accessibility in the sensory progenitors (μATACseq, MACS2, IDR<0.1). Heatmaps also indicate the presence of
H3K4me1, and ATOH1 binding at the 18,681 hair cell-enriched distal elements. Dashed line separates the ATOH1
targets from non-ATOH1 targets in each group (defined by IDR< 0.1, from two biological replicates of ATOH1 C&R).
(D) Left: Motif enrichment analysis of the 2,904 pre-established ATOH1 targets. Middle: Gene ontology results
associated with the pre-established ATOH1 targets suggest that enhancers for Notch signaling components are
poised to be activated in the sensory progenitors. Right: The Hes6 locus serves as an example of the 2,904 pre-
established ATOH1 targets, which are already open and H3K4me1-labeled in the sensory progenitors. (E) Left: Motif
enrichment analysis of the 3,264 de novo ATOH1 targets suggests that POU domain transcription factors maybe
more involved in the de novo ATOH1 targets, compared to the pre-established ones. Middle: Gene ontology
associated with the 3,264 de novo ATOH1 targets suggests many enhancers for hair cell differentiation and
mechanosensation are only created in the hair cells in a de novo fashion. Right: The Gfi1 locus serves as an example
of the 3,264 de novo ATOH1 targets, which are not open or labeled by active histone modifications in the sensory
progenitor cells.
24
accessible (FDR < 0.1; Figure 1B), and an additional 24,460 distal elements were not
significantly changed during the transition from E13.5 progenitors to E17.5 hair cells.
We focused our analysis on the hair cell-enriched distal elements, as their gain in
accessibility suggests their potential in regulating important changes in gene expression
during the developmental transition (Frank et al., 2015; Klemm et al., 2019; Mueller et
al., 2017). Of the 18,681 distal elements that were significantly enriched in E17.5
nascent hair cells, around 60% (11,133) were nonetheless discernible (peak IDR < 0.1)
in undifferentiated E13.5 sensory progenitors (Figure 1C), suggesting that these pre-
established elements are potential enhancers, which are already primed for activation.
To test this, we used CUT&RUN (C&R) and found that these elements contain
nucleosomes marked by H3K4me1 (Figure 1C), a marker for primed enhancers (Calo
and Wysocka, 2013; Creyghton et al., 2010), in the undifferentiated progenitors. In
addition, these 11,133 pre-established chromatin regions include 2,904 distal elements
that become targets of ATOH1 when it is expressed in nascent hair cells, as shown by
ATOH1 C&R (IDR<0.1, Figure 1C). Several of these elements are associated with
genes of the Notch signaling pathway in hair cells, such as Jag2, Dll1, Mfng, Hes6
(Figure 1D).
While we found that around 60% of distal elements enriched in hair cells were pre-
established in the progenitor population, the remaining ~40% (7,548 / 18,681) of hair
cell enriched distal elements were not accessible in sensory progenitors, and appeared
de novo in nascent hair cells (Figure 1C, bottom panel). Moreover, these elements were

25

not labeled by H3K4me1 or other histone modifications (H3K27ac, H3K4me3 and
H3K27me3; Figure 1C and Figure 3A, B) in the progenitors. This suggests these distal
elements are silent in sensory progenitors, but become accessible and primed as hair
cell differentiation is induced. Of these 7,548 distal elements, we identified 3,264
elements as direct ATOH1 targets by C&R (IDR <0.1, Figure 1C). This group of de novo
ATOH1 targets, which are activated in differentiating hair cells, contain many putative
enhancers near genes responsible for hair cell maturation and the elaboration of the
mechanosensory machinery, such as Gfi1 (Figure 1E), Lhx3, Tmc1, Insc, Lhfpl5, and
Myo6.
Figure 2. Correlation analysis at the
promoters and distal elements
detected in the sensory progenitors
and hair cells. (A) Scatter plot shows
that the ATACseq data at the promoter
regions between sensory progenitors
and hair cell are highly correlated. (B)
Heatmap representation of the
ATACseq results at the promoter
regions in sensory progenitors and hair
cells. Similar promoter accessibility
could be observed between hair cell
and progenitors even at genes with
very different expression levels. (C)
Scatter plot shows the Pearson
correlation of the ATACseq data at the
distal elements between sensory
progenitors and hair cells. (D)
Heatmap representation of the
ATACseq results at the distal elements
in sensory progenitors and hair cells.
Three groups of the distal elements are
defined in Figure 1B.

26
POU4F3 is necessary to provide ATOH1 access to its targetome in nascent hair
cells.
We next asked how ATOH1 is able to recognize its targets in inaccessible chromatin
regions during the transition from sensory progenitors to differentiating hair cells.
Structural analysis suggests that most bHLH transcription factors, including ATOH1,
contain an extended recognition α-helix in the basic domain that limits their ability to
bind nucleosome-occupied DNA (Fernandez Garcia et al., 2019; Soufi et al., 2015).
Therefore, we hypothesized that other hair cell-specific transcription factors may be
required to act as pioneer factors to overcome nucleosomal barriers and allow ATOH1
to fully engage its targets. Indeed, we observed that the vast majority of the de novo
distal elements lacked any of the tested, active or repressive, histone modifications in
sensory progenitors (Figure 3). This is reminiscent of the requirements for pioneer factor
activity during somatic cell reprogramming to pluripotency (Soufi et al., 2015). We
performed motif enrichment analysis on all identified ATOH1 target elements, and found
that POU-domain transcription factor family motifs were more highly enriched in the de
novo ATOH1 targets that become accessible in hair cells, compared to pre-established
ATOH1 targets that are already accessible in sensory progenitors (Figure 1E versus
1D).
POU4F3 is an excellent candidate to act as a pioneer factor in hair cell
differentiation, as it is the only POU-domain transcription factor highly expressed in hair
cells (FPKM > 400). We therefore tested whether POU4F3 is necessary for the
acquisition of chromatin accessibility at de novo ATOH1 targets in differentiating hair
27
cells. Previous studies have
shown that hair cells begin to
develop normally in the
absence of Pou4f3, but then
die rapidly after birth
(Hertzano et al., 2004; Xiang
et al., 1998). We used Atoh1-
Gfp reporter mice (Rose et al.,
2009) to show that ATOH1-
GFP (fusion protein) is still
highly expressed in four rows of
MYO7A-positive hair cells in
Pou4f3 mutant cochleae at
E17.5 (Figure 4A, B), confirming that ATOH1 expression is not dependent on POU4F3,
and enabling us to FACS-purify and analyze GFP+ hair cells from wild type and Pou4f3
mutant cochleae at E17.5. Quantitative analysis of μATACseq profiles revealed that
around 45% (1,458 / 3,264) of the de novo ATOH1 targets were significantly less
accessible in Pou4f3 mutant hair cells (POU4F3-dependent ATOH1 targets; FDR < 0.1,
fold change > 2; Figure 4C). C&R profiling confirmed that POU4F3 preferentially bound
to these sites (Figure 4D), and is thus likely to directly regulate their chromatin
accessibility. In addition to the reduced chromatin accessibility, ATOH1 binding at these
1,458 POU4F3-dependent ATOH1 targets was dramatically reduced in Pou4f3 mutant
Figure 3. The de novo hair cell-enriched distal elements are not
marked by any tested histone modifications. (A) Heatmap
representation of the ATACseq and CUT&RUN results of the active
histone marks at the hair cell-enriched distal elements. (B) Scatter plot
representation of the CUT&RUN results of the repressive histone mark
H3K27me3 at the hair cell-enriched distal elements
28
Figure 4. POU4F3 is necessary to provide ATOH1 access to its complete targetome. (A and B) Confocal images of
the E17.5 cochlea (whole mount and cryo-section immunohistochemical staining) show ATOH1-GFP is highly
expressed in 4 rows of MYO7A+ hair cells in both wild type and Pou4f3-mutant hair cells, confirming that the early
specification of hair cells is not dependent on the expression of Pou4f3. Scale bar: 20 μm. (C) Heatmap
representation of μATACseq results at the 3,264 de novo ATOH1 targets, from sensory progenitors, wild type hair
cells and Pou4f3-mutant hair cells. These distal elements are clustered into two groups based on chromatin
accessibility indicating a large group (1,458) of POU4F3-dependent loci (n=2; False discovery rate <0.1); (D)
Heatmap representation of C&R results for POU4F3 in wild type hair cells, indicating preferential binding of POU4F3
to the POU4F3-dependent ATOH1 targets; (E) Heatmap representations of the C&R results for ATOH1 in wild type
and Pou4f3-mutant hair cells, indicating that the loss of ATOH1 binding at these sites correlates with loss of Pou4f3
expression and loss of chromatin accessibility (C, μATACseq); (F) Heatmap representations of the C&R results for
H3K4me1 and H3K27ac in wild type and Pou4f3-mutant hair cells, indicating that acquisition of active enhancer
marks at POU4F3-dependent sites is correlated with Pou4f3 expression, and chromatin accessibility (μATACseq).
(G) Scatter plot and box plot representations of the RNAseq comparison between wild type and Pou4f3-mutant hair
cells. Red dots represent hair cell-specific genes within 50kb of POU4F3-dependent ATOH1 targets. Green dots
represent the hair cell-specific genes within 50kb of POU4F3-independent ATOH1 targets. Blue dots represent the
genes that are not significantly changed. Box plot insert with median and quartile statistics, indicating that genes
associated with POU4F3-dependent ATOH1 targets are specifically affected by the loss ofPou4f3 expression. (H)
Representative gene tracks showing the μATACseq and C&R data at the putative enhancers for two deafness-
related genes, Myo3b and Slc8a2, illustrating enhancer changes at genes whose expression is reduced in response
to loss of POU4F3 binding at ATOH1 targets
29
hair cells (Figure 4E). In contrast, the binding of ATOH1 to the other 1,806 POU4F3-
independent de novo ATOH1 targets was far less affected in Pou4f3 mutant hair cells
(Figure 4E). These results indicate that POU4F3 binds nucleosomal DNA, and is
necessary for the chromatin rearrangement that occurs in
~
45% of ATOH1 targets in the
transition from E13.5 progenitors to hair cells at E17.5.
To investigate whether changes in gene expression occurred preferentially at
POU4F3-dependent ATOH1 targets in Pou4f3 mutant hair cells, we compared the
transcriptome of E17.5 hair cells from wild type and Pou4f3 mutant cochleae by
RNAseq (Figure 4G). We focused our analysis on the 656 genes that were highly
upregulated during the transition from progenitors to nascent hair cells (E17.5 hair cells
versus E13.5 progenitors, fold change > 4, FDR <0.1, FPKM >5), and which lay within
50,000 bp of the 3,264 de novo ATOH1 targets. The expression of most of these hair
cell-specific genes was significantly reduced in Pou4f3 mutant hair cells (Figure 4G).
Notably, genes associated with POU4F3-dependent ATOH1 targets were predominately
reduced in Pou4f3 mutants (p < 0.001, Figure 4D, red box), including many genes
related to mechanotransduction and human deafness (e.g. Grxcr1, Atp8a2, Adcy1,
Ush2a, Myo3b, Piezo2) (Figure 4G).
The reduced expression of ATOH1 target genes in Pou4f3 mutant hair cells could be
a consequence of the loss of enhancer activity at these POU4F3-dependent targets. We
tested this by profiling active histone modifications at these elements (H3K4me1 and
H3K27ac). We found the levels of H3K4me1 and H3K27ac at the POU4F3-dependent
ATOH1-targets were significantly reduced in Pou4f3 mutants, whereas the levels of
30
these marks of active enhancers remained largely unchanged at POU4F3-independent
ATOH1 target elements (Figure 4F and 4H).
Taken together, these results indicate that POU4F3 is necessary for accessibility of
~
45% of de novo ATOH1 targets in nascent hair cells, and required for ATOH1 binding
to these sites. In the absence of POU4F3 to open these distal elements, the associated
ATOH1 target gene expression is significantly reduced.

Figure 5. POU4F3 binds closed chromatin and facilitates ATOH1 binding at many ATOH1-targets in a
heterologous cell type. (A) Heatmap representation of the μATACseq landscape at the 1,458 POU4F3-dependent
ATOH1 targets identified in Figure 2C. Data from viral overexpression of GFP (control), ATOH1 (ATOH1-OE),
POU4F3 (POU4F3-OE) in MEFs, was compared to μATACseq from wild type hair cells and Pou4f3-mutant hair cells
collected at E17.5 (provided for comparison; from Figure 2C). The 1,458 distal elements that were dependent on
Pou4f3 expression for chromatin accessibility in hair cells were re-clustered on the basis of their chromatin
accessibility in the infected MEFs. Cluster 1 (1,196): regions remaining closed in ATOH1, POU4F3 or GFP
overexpressed MEF; Cluster 2 (131): regions induced by overexpression of POU4F3 alone; Cluster 3 (103): regions
induced by overexpression of ATOH1 alone; Cluster 4 (28): Regions accessible in control. (B) Upper panel: Heatmap
representations of μATACseq results of MEFs overexpressed with ATOH1 and POU4F3 together, at the 1,196
regions that remain closed in overexpression of individual transcription factors (Group 1 in A). Results show that a
small portion of these regions (233) were made accessible by the synergy between POU4F3 and ATOH1. However,
the majority remained closed. Lower panel: Heatmap representations and the averaged signal profiles of the C&R
results for ATOH1 and POU4F3 in the reprogrammed MEFs, at the 963 distal elements that remain closed in ATOH1
and POU4F3 co-overexpressed MEF. Results show that POU4F3 is able to bind to closed chromatin at these regions
when overexpressed alone. ATOH1 cannot bind to closed chromatin by itself, but can bind when co-overexpressed
with POU4F3, suggesting POU4F3 facilitates the binding of ATOH1, through protein-protein interaction or through the
POU4F3-induced changes of chromatin conformation. However, these regions remain closed, even when co-bound
by ATOH1 and POU4F3, suggesting other hair cell-specific transcription factors are required to facilitate chromatin
accessibility.
31
POU4F3 binds closed chromatin in a heterologous cell type, and can synergize
with ATOH1 to stimulate open chromatin formation.
Previous unbiased screening for the nucleosome binding ability of transcription factors
predicts that several POU-domain transcription factors have strong nucleosome binding
potential (Fernandez Garcia et al., 2019). Therefore, we sought to determine if POU4F3
is associated with pioneer factor activity at previously identified ATOH1 target elements
that require POU4F3 for their accessibility in hair cells (Figure 4C). We overexpressed
ATOH1 and POU4F3, alone and together in mouse embryonic fibroblasts (MEFs),
followed by μATACseq to assess chromatin structure changes at the 1,458 POU4F3-
dependent ATOH1 targets that we identified in hair cells (Figure 4C and Figure 5A).
We grouped these 1,458 hair cell elements based on their µATACseq accessibility in
MEFs into four groups (Figure 5A). Although expressing POU4F3 or ATOH1 alone was
able to “open” a small number of these sites (103 sites by ATOH1; 131 sites by
POU4F3; and 28 sites in Control) (Figure 5A), presumably through collaboration with
co-factors present in MEFs, a large majority of POU4F3-dependent ATOH1 targets
(1,196/1,458) remained closed in MEFs (Figure 5A). Among these 1,196 sites that failed
to open in response to ATOH1 or POU4F3 alone, some sites (233) became accessible
when ATOH1 and POU4f3 were overexpressed together in MEFs (Figure 5B, Group 1),
suggesting a synergy between these two factors. However, 963 remained closed,
suggesting that additional factors not present in MEFs are needed to induce chromatin
rearrangement at these sites. Motif enrichment analysis indicated that in addition to
POU4F3 and ATOH1 cognate motifs, these 963 regions were also enriched for LHX
32
and GATA transcription factor motifs (Figure 5B, upper panel). Consistent with this,
studies from our lab, and others, have shown that Lhx3, Gata3 (Bardhan et al., 2019;
Hertzano et al., 2007; Luo et al., 2013; Menendez et al., 2020) are expressed in nascent
hair cells, but that these factors are not expressed in MEFs (Menendez et al., 2020).
To test whether POU4F3 has the ability to bind closed chromatin in this heterologous
cell type, we analyzed ATOH1 and POU4F3’s ability to bind closed chromatin at the
remaining 963 chromatin regions using C&R (Figure 5B, upper panel, group 2), whose
accessibility was not induced by the co-expression of ATOH1 and POU4F3 together.
POU4F3, when exogenously expressed in MEFs, was able to bind closed chromatin at
these sites (Figure 5B, lower panel), demonstrating a key characteristic of pioneer factor
activity (Iwafuchi-Doi and Zaret, 2014, 2016). In contrast, lentivirus-expressed ATOH1-
alone was unable to bind to these sites in MEFs (Figure 5B, lower panel). Interestingly,
ATOH1 and POU4F3 were able to synergize and allow ATOH1 binding, although the
pair remained unable to induce chromatin accessibility, based on the μATACseq profile
(Figure 5B, upper panel). Together, these data show that POU4F3 is able to bind to
closed chromatin, while ATOH1 does not. However, POU4F3 and ATOH1 can
synergize to allow ATOH1 binding in closed chromatin.
ATOH1 and POU4F3 synergize to form a feed-forward regulatory circuit in
differentiating hair cells
Our data, described in Figure 4, demonstrate that POU4F3 is required to open
chromatin associated with many distal elements that are bound by ATOH1. However,
Pou4f3 mRNA has been reported to be expressed in the cochlea later than ATOH1
33
(Ahmed et al., 2012; Pan et al., 2012). To help explain this seeming paradox, we first
confirmed that Atoh1 expression precedes Pou4f3 in the developing cochlea. Hair cell
differentiation in the cochlea is known to propagate in a basal-to-apical gradient
(Bermingham et al., 1999; Cai et al., 2013; Chen et al., 2002). At E15.5, we found that
ATOH1 and POU4F3 proteins co-localized in four rows of hair cells at the base of the
cochlea (Figure 6A). However, in the less differentiated apex, only ATOH1 could be
detected in a group of progenitors (Figure 6A). Moreover, when we used a Pax2-Cre
transgenic mouse (Ohyama and Groves, 2004) to conditionally delete Atoh1 starting at
E8.5, we were unable to detect POU4F3 protein in the E15.5 cochlear sensory epithelia
(Figure 6B). Together these data suggest that ATOH1 expression precedes and is
necessary for the expression of Pou4f3 in differentiating hair cells.
Our data confirm that Atoh1 is epistatic to Pou4f3 (Figure 6A, B), and show that
POU4F3 is required for the accessibility of
~
45% of the ATOH1 binding sites that appear
de novo at the time of Atoh1-upregulation in E13.5 progenitors (Figure 4C). Moreover,
we show that POU4F3 can bind to closed chromatin and enable the sequential binding
of ATOH1 (Figure 5A, B). These observations invite the question of how these “closed”
sites become accessible to ATOH1. To answer this, we hypothesized a model in which
an epigenetic feed-forward circuit is initiated by the direct binding of ATOH1 to a pre-
existing open enhancer in sensory progenitors near the Pou4f3 locus. This binding of
ATOH1 is sufficient to stimulate POU4F3 expression, and initiate pioneer factor activity
at downstream ATOH1 targets. Consistent with this model, µATACseq and C&R results
showed that ATOH1 directly bound to several accessible, hair cell-enriched distal
34
Figure 6. ATOH1 and POU4F3 synergize to form a feed-forward regulatory circuit in differentiating hair cells. (A) Left:
Confocal image of a mouse cochlea at E15.5 (whole mount immunohistochemistry). The red dashed rectangle
represents an apical region, and the blue dashed rectangle represents a middle-basal region of the developing
cochlea. Scale bar: 100 μm. The differentiation of the organ of Corti follows a basal-to-apical gradient. Therefore at
any given developmental stage, the base of the cochlea is always more developed compared to the apex. Right:
Enlarged view of the confocal images, corresponding to the apical and the middle-basal regions of the cochlea shown
in (left). At the mid-base, the expression of ATOH1 and POU4F3 co-localize in four rows of hair cells. In contrast, at
the less differentiated apex, only ATOH1 can be detected in a group of SOX2-positive progenitors, suggesting the
expression of Atoh1precedes that of Pou4f3. Scale bar: 20 μm. (B) Confocal images of the middle-basal region of the
mouse cochlea at E15.5, from both wild type and Atoh1 conditional knockout mice using Pax2-Cre. No hair cell is
formed in Atoh1 conditional knockout cochlea. No POU4F3 is detected in the Atoh1 conditional knockout cochlea.
Scale bar: 20 μm. (C) Genome browser representation of the μATACseq and C&R results at the Pou4f3 locus. The
black arrows are the putative ATOH1-bound enhancers for Pou4f3 upregulation in the hair cells. ATOH1 binds to
several pre-established enhancers (accessible and labeled by H3K4me1) in the progenitor cells. And the binding of
ATOH1 to these regions is not affected by the absence of Pou4f3 expression. These data suggest that Pou4f3
upregulation in the hair cells is directly controlled by ATOH1. (D) Confocal images of the in vitro cultured utricles from
Atoh1 conditional knockout mice. The utricles are overexpressed with GFP (control), or ATOH1-GFP, encoded in the
Adeno-associated viruses (AAV). Overexpression of ATOH1, not GFP, induced the expression of POU4F3,
suggesting that ATOH1 is sufficient for Pou4f3 expression in the inner ear. Scale bar: 20 μm.
35
elements upstream of the Pou4f3 promoter in a POU4F3-independent manner (Figure
6C), including one site (Figure 6C; enhancer 3), which has previously been shown to be
a bona fide hair cell-specific Pou4f3 enhancer (Masuda et al., 2011). Analysis of
accessibility and H3K4me1 marks showed that two of these distal elements (Figure 6C;
enhancer 1 and 3) were accessible and poised (H3K4me1-positive) (Calo and
Wysocka, 2013; Creyghton et al., 2010) in E13.5 progenitors, prior to Pou4f3
expression. These data suggest that the initial up-regulation of Pou4f3 in the developing
cochlea is directly controlled by ATOH1.
We next tested if ATOH1 overexpression is sufficient to drive Pou4f3 expression in the
inner ear. We generated AAV2/ Anc80L65 viruses (Suzuki et al., 2017) expressing
ATOH1-t2a-GFP or GFP alone, and used these to infect inner ear tissue from Atoh1
mutant mice. Since cochlear sensory progenitors undergo apoptosis in the absence of
ATOH1 (Cai et al., 2013; Chen et al., 2002), we over-expressed ATOH1 in the Atoh1
mutant utricle, a vestibular sensory organ in the inner ear containing hair cells. Unlike
the cochlea, the utricular sensory epithelium in Atoh1 mutant mice does not degenerate
embryonically, but remains as a monolayer of SOX2-positive progenitor cells with a
normal morphology (Bermingham et al., 1999). Re-introducing ATOH1 into Atoh1
mutant tissues induced expression of POU4F3 (Figure 6D), as well as other hair cell-
specific protein including MYO7A and LHX3 (data not shown), indicating that sensory
epithelia from Atoh1 mutant utricles retain their potential to express Pou4f3 and
differentiate into hair cells. Taken together, these data demonstrate that Pou4f3 is a
direct downstream target of ATOH1 in differentiating hair cells, and is part of a feed-
36
forward mechanism that is recruited by ATOH1 to gain access to its 1,458 de novo
targets deployed in differentiating hair cells.
Figure 7. The ATOH1-POU4F3 feed-forward circuit is conserved in mechanosensory Merkel cells. (A) Confocal
images of the touch domes from wild type and Pou4f3-mutant hairy skin. The expression of early Merkel cell markers
(ATOH1 and SOX2) in the touch dome is not dependent on Pou4f3 expression. However, the mature Merkel cell
marker KRT20 is not expressed in Pou4f3 mutant Merkel cells, suggesting that like nascent hair cells, Merkel cells
are specified in the absence of Pou4f3, but cannot develop further. Scale bar: 40 μm. (B) Quantitative comparison of
the chromatin accessibility at the distal elements between the touch dome epithelial progenitors and the Merkel cells
(two μATACseq biological replicates for each cell type, false discovery rate < 0.1). 28,461 distal elements are
significantly more open in the differentiating Merkel cells (Merkel cell-enriched distal elements). (C) Heatmap
representation of the chromatin accessibility, the profile ATOH1 binding at the 28,461 Merkel cell-enriched distal
elements, which are clustered into two groups based on their accessibility in the touch dome epithelial progenitors
(open distal elements in the progenitors are defined by irreproducible discovery rate < 0.1, from two biological
replicates of μATACseq). Dashed line separates the ATOH1 targets from non-ATOH1 targets in each group (ATOH1
targets in the Merkel cells are defined by irreproducible discovery rate < 0.1, from two biological replicates of C&R).
Like in the hair cells, ATOH1 also binds to pre-established and de novo distal elements in Merkel cells. (D) Left:
Heatmap representation of the μATACseq results at the 4,683 de novo ATOH1 targets, from touch dome epithelial
progenitors, wild type Merkel cells and Pou4f3-mutant Merkel cells. These distal elements are clustered into two
groups based on whether the chromatin accessibility are significantly reduced in Pou4f3-mutant Merkel cells,
compared to wild type Merkel cells (False discovery rate <0.1, DESeq2). Right: Heatmap representation of the C&R
results for POU4F3 in wild type Merkel cells suggest POU4F3 directly regulate the chromatin accessibility at these
POU4F3-dependent ATOH1 targets in Merkel cells. (E) Genome browser representations of the μATACseq and C&R
data at two POU4F3-dependent ATOH1 targets in Merkel cells.
37
The ATOH1-POU4F3 feed-forward circuit is conserved in Merkel cells
Merkel cells are a population of epidermally-derived secondary receptor cells
specialized for tactile discrimination and the sensation of light touch (Ikeda et al., 2014;
Maksimovic et al., 2014; Woo et al., 2014b). Merkel cells and sensory hair cells share
an ectodermal origin, but their lineages diverge in early in embryogenesis, with Merkel
cells deriving from non-neural ectoderm, while sensory hair cells derive entirely from the
otic placode (Groves et al., 2013; Morrison et al., 2009; Van Keymeulen et al., 2009).
Despite their different embryonic origins, and their division of labor in perceiving
different external mechanical stimuli, hair cells and Merkel cells share cellular and
molecular features (Lumpkin and Caterina, 2007), and are suggested to be “sister” cell
types, derived from an ur-mechanoreceptor (Arendt et al., 2016). Notably, Merkel cells
also express ATOH1 and POU4F3, and require ATOH1 for their differentiation (Maricich
et al., 2009b). Immunostaining of developing touch domes, in the epidermis of Pou4f3
mutant mice also containing an Atoh1-Gfp reporter, show that Merkel cells lacking
POU4F3 still express the early Merkel cell markers ATOH1 and SOX2 (Figure 7A)
(Laga et al., 2010), but not KRT20, a more mature Merkel cell keratin (Figure 7A)
(Perdigoto et al., 2014). This suggests that, like nascent hair cells, Merkel cells are
specified in the absence of Pou4f3, but cannot complete their program of differentiation.
Thus, the feed-forward role of POU4F3 seen in hair cells may also be important in the
maturation of Merkel cells.
These findings prompted us to investigate whether POU4F3 also regulates the
accessibility of ATOH1 targets in a feed-forward manner during the maturation of Merkel
38
cells. Merkel cell progenitors (touch dome epithelial progenitors, TDEP) reside among
the epithelial precursors of touch domes in E17.5 epidermis, and can be isolated by
FACS-purification using a unique combination of expressed antigens (Integrin-α6,
Cd34, Sca-1, and CD200; Doucet et al., 2013); whereas differentiating Merkel cells can
be isolated by their expression of ATOH1-GFP. We used Atoh1-Gfp mice and Atoh1-
Gfp; Pou4f3 mutant mice to FACS-purify touch dome epithelial progenitors and Merkel
cells from wild type and Pou4f3 mutant epidermis. We analyzed accessible chromatin
with μATACseq and identified 28,461 out of a combined 64,658 open distal elements
that were enriched in E17.5 Merkel cells, compared to touch dome epithelial precursors
(DESeq2, FDR<0.1, Figure 7B), while 24,355 elements were more accessible in touch
dome epithelial precursors, and 11,842 distal elements remained unchanged during the
differentiation process.
We focused our analysis on the 28,461 Merkel cell-enriched distal elements, and
found that a small portion of these were already present (pre-established) in the TDEPs
(1,756/28,461), while the majority arose de novo during Merkel cell differentiation
(26,696/28,461) (Figure 7C), among which 4,683 (IDR<0.1) were highly enriched for
ATOH1 binding by C&R (Figure 7C). To assay the number of these de novo ATOH1
targets that are dependent on POU4F3 for accessibility, we compared their µATACseq
profile between wild type and Pou4f3 mutant Merkel cells. We found that the
accessibility of 710 of these 4,683 de novo ATOH1 target elements was dependent on
the expression of POU4F3, and that these 710 elements bound POU4F3 by C&R
(Figure 7D). Many of these POU4F3-dependent de novo ATOH1 target elements are
39
near genes critical for Merkel cell maturation and mechanotransduction, such as Krt20,
Rab3c (Haeberle et al., 2004; Perdigoto et al., 2014; Figure 7E), and the cardinal light-
touch mechano-transduction channel Piezo2 (Ikeda et al., 2014; Maksimovic et al.,
2014; Ranade et al., 2014; Woo et al., 2014b) (Figure 8D). RNAseq confirmed that
expression of Piezo2, Rab3c and Krt20 was significantly reduced in Pou4f3 mutant
Merkel cells (FDR < 0.1). Together, these results suggest that, just as in hair cells, the
pioneer factor activity of POU4F3 is required for ATOH1 to correctly coordinate
mechanosensory differentiation in Merkel cells.
The ATOH1-POU4F3 synergy is conserved between mechanosensory cell types
with divergent enhancer networks
It has been suggested that sensory hair cells and Merkel cells arose evolutionarily from
a common mechanoreceptor cell type (Arendt et al., 2016). One mechanism for such a
divergence would be the appearance of unique enhancer targets in each sister cell that
depend on ATOH1/POU4F3 synergy to potentiate a pool of regulatory elements that
contain the motifs of both transcription factors, but ultimately diverge in their utilization.
To test this, we compared the accessibility of POU4F3-dependent ATOH1 targets
identified in hair cell and Merkel cells, and found 833 distal elements that were
commonly accessible (μATACseq; Figure 8A, B). C&R analysis showed that POU4F3
and ATOH1 bound to these common regions in both hair cells and Merkel cells (Figure
8B). Gene ontology (GO) analysis showed that these common regulatory elements are
40
Figure 8. The ATOH1-POU4F3 synergy represents a conserved gene regulatory mechanism for differentiation of
mechanosensory cells with divergent enhancer networks. (A) Venn diagram shows the comparison of chromatin
accessibility at all the POU4F3-dependent ATOH1 targets identified from hair cells and Merkel cells. We combined
the 1,458 hair cell-specific POU4F3-dependent ATOH1 binding sites in Figure 4, and the 710 Merkel cell-specific
POU4F3-dependnet ATOH1 binding sites in Figure 7. And we asked which distal elements are only open in Merkel
cells (523), commonly open in both cell types (833), and which one is only open in hair cells (683). (B) Heatmap
shows the chromatin accessibility, and the binding profile of POU4F3 and ATOH1 at the three groups of the POU4F3-
dependent ATOH1 targets revealed in (A). POU4F3 is able to weakly bind to Merkel cell-specific sites in hair cells,
which are closed in hair cells, and vise versa, consistent with its pioneer factor activity. (C) Motif enrichment analysis
of the three groups of the POU4F3-dependent ATOH1 targets revealed in (A). Different sets of transcription factor
motifs are enriched in Merkel cell-specific and hair cell-specific POU4F3-dependent ATOH1 targets, suggesting a
context-dependent specificity of the opening and activation of POU4F3-dependent ATOH1 targets in different cell
types. (D) Genome browser representation of the μATACseq and C&R data at examples of the POU4F3-dependent
ATOH1-targets that are Merkel cell-specific, common, and hair cell-specific.
41
enriched near genes responsible for synaptogenesis and ion channels (Figure 9A). For
example, the PIEZO2 channel protein is present in both Merkel cells and hair cells.
While PIEZO2 is crucial for Merkel cell mechanosensitivity, it nonetheless contributes to
the function of both cell types (Ikeda et al., 2014; Maksimovic et al., 2014; Ranade et
al., 2014; Woo et al., 2014b; Wu et al., 2017). We identified a potential intronic
enhancer in the Piezo2 locus (H3K27ac+ and H3K4me1+ in hair cells, data not shown),
that was bound by ATOH1 and POU4F3 in both hair cells and Merkel cells (Figure 8D),
and whose chromatin was not accessible in either cell type in Pou4f3 mutant mice
(Figure 8D). This suggests that the ATOH1/POU4F3 feed-forward circuit may affect
Piezo2 expression in both mechanosensory cell types by regulating accessibility and
activity at the same regulatory element.

Figure 9. Gene ontology analysis of the POU4F3-dependent ATOH1 targets in hair cells and
Merkel cells. (A) Go term results from Jensen COMPARTMENTS database of the POU4F3-
dependent ATOH1 targets in hair cells and Merkel cells. (B) Go term results from GREAT
Components database of the POU4F3-dependent ATOH1 targets in hair cells.
42

In contrast to the ATOH1/POU4F3 targets that are in common between hair cells and
Merkel cells, we also found that 523 regions were only open in Merkel cells, and that
683 regions were only open in hair cells (Figure 8A, B). C&R analysis showed that
POU4F3 and ATOH1 binding also diverged in a cell type-specific manner (Figure 8B
and 8D). Among the 683 hair cell-specific POU4F3-dependent ATOH1 targets, many
were near genes encoding unique components of the hair cell mechanotransduction
machinery (Figure 8D, Figure 9), (e.g. Grxcr1, Lhfpl5, Ush2a, Clrn1, Cdh23, Myo3b)
(Pepermans and Petit, 2015; Petit and Richardson, 2009). GO terms associated with
these distal elements included “stereocilium”, and “cluster of actin-based cell
projections” (Figure 9A, B), while the 523 putative regulatory elements enriched in
Merkel cells generated GO terms such as glutamatergic synapses, integrin complex and
inward rectifier potassium channel complex (Figure 9A). Motif enrichment analysis
revealed that the binding sites for SIX and GATA transcription factor family members
were differentially represented among the 683 hair cell-specific POU4F3-dependent
ATOH1 targets, compared to those in Merkel cells (Figure 8C). Immunostaining
confirmed that SIX1 was broadly expressed in the cochlea at E13.5 and E17.5 (Figure
10A, B) (Ahmed et al., 2012; Zhang et al., 2017), as is GATA3 (Bardhan et al., 2019;
Luo et al., 2013). In contrast, TFAP2 and SOX-factor motifs were differentially enriched
in the Merkel-cell specific sites (Figure 8C), and we found that TFAP2A is expressed in
Merkel cells, as well as the epidermal cells within and surrounding the touch domes
43
(Figure 7A) (Li et al., 2019). No additional motifs, beyond POU4F3 and ATOH1, were
highly enriched at those sites in common (833) between the two cell types (Figure 8C).
Interestingly, POU4F3 seems to bind weakly to many of the Merkel cell-specific
targets that lie in closed chromatin in hair cells, and vice versa (Figure 8B), consistent
with the ability of POU4f3 to bind to closed chromatin. Taken together, we propose that
the ATOH1/POU4F3 feed-forward circuit may constitute part of the core gene regulatory
mechanism for mechanosensation, which is complemented by distinct enhancer
networks that are regulated by lineage-specific transcription factors to allow the
differentiation of specific mechanosensory cell types.

Figure 10. Six1 is broadly
expressed in the cochlea. (A)
Confocal images of the E13.5
cochlea. Six1 is highly
expressed in every cell in the
cochlea. Scale bar: 20 μm. (B)
Confocal images of the E17.5
cochlea. Six1 is highly
expressed in hair cells and
supporting cells, as well as
other cells in the cochlea.
Scale bar: 20 μm.

44
DISCUSSION
During development and regeneration, the correct coordination of progenitor cell
differentiation relies on changing patterns of gene expression, which in turn rely on
specific patterns of chromatin accessibility and epigenetic modifications (Cantone and
Fisher, 2013; Klemm et al., 2019). However, the mechanisms that regulate these
hierarchical changes in chromatin structure are poorly understood. We used the
differentiation of sensory hair cells in the developing inner ear to show that a significant
number of chromatin targets of the bHLH transcription factor ATOH1, which is
necessary and sufficient for hair cell differentiation in the context of the inner ear
(Bermingham et al., 1999; Zheng and Gao, 2000), are located in inaccessible chromatin
when Atoh1 is first up-regulated in undifferentiated cochlear sensory progenitors (Figure
1C). We describe a feed-forward synergy between ATOH1 and a downstream
transcription factor POU4F3, in which ATOH1 first stimulates the expression of Pou4f3
in differentiating hair cells, with POU4F3 then making additional ATOH1 targets
accessible to ATOH1 binding. We show that POU4F3 has the ability to bind “closed”
chromatin (Figure 5), and to stimulate chromatin accessibility in synergy with ATOH1
and other co-factors in the manner of a classic pioneer factor (Iwafuchi-Doi and Zaret,
2014, 2014; Soufi et al., 2015). Remarkably, this feed-forward mechanism governing
sequential gene expression in hair cells is conserved in another mechanosensory cell
type, Merkel cells. We hypothesize that this feed-forward mechanism arose in an
ancestral neurosensory mechanoreceptor cell type, and may have provided a basis for
45
sister-cell evolution through enhancer network divergence between modern
mechanoreceptors (Arendt, et al., 2016).
Feed-forward synergy between ATOH1 and POU4F3 is necessary for hair cell
development
We present several lines of evidence for the feed-forward synergy between ATOH1 and
POU4F3. First, our μATACseq analysis of hair cell progenitors shows that over 50% of
ATOH1 binding sites in hair cells are in an epigenetically inaccessible state (3,264 de
novo ATOH1 targets vs 2,904 pre-established ATOH1 targets, Figure 1C) in hair cell
progenitors, and cannot be bound by ATOH1, when ATOH1 is expressed in a
heterologous cell type (mouse embryonic fibroblasts, MEFs) (Figure 5B). This implies
that additional mechanisms must, by necessity, be deployed to make these binding
sites accessible, before further steps in hair cell development can proceed. Second, we
show these distal elements that are inaccessible to ATOH1, are enriched for POU factor
binding sites (Figure 1E), and that POU4F3 is able to bind these elements when over-
expressed in MEFs where ATOH1 is unable to bind them (Figure 5B). Third, our
analysis of Pou4f3 null mice shows that POU4F3 binding is necessary for these
elements to become accessible (Figure 4C), and for the hair cell genes associated with
them to be transcribed (Figure 4D). Finally, we show that Atoh1 is required for Pou4f3
expression, and that Pou4f3 is a direct transcriptional target of ATOH1 (Figure 6). After
ATOH1 levels begin to rise in differentiating hair cells, POU4F3 levels rise sufficiently to
stimulate the pioneer factor activity needed to make additional hair cell loci accessible,
allowing ATOH1 to continue driving the differentiation of hair cells, and making
46
contingent the transition from E13.5 progenitor to nascent hair cell, providing a
mechanistic basis for the observed epistasis between Atoh1 and Pou4f3.
We speculate that this feed-forward synergy between ATOH1 and POU4F3
contributes to the precise and stereotyped arrangement of hair cells and supporting
cells in the organ of Corti that is essential for the ability of the cochlea to detect sound.
Lateral inhibitory Notch signaling from hair cells to surrounding cells is one mechanism
that has been shown to contribute this precise pattern (Abdolazimi et al., 2016; Kiernan,
2005; Lanford et al., 1999; Tateya et al., 2011). In addition, lineage tracing experiments
with Atoh1-Cre and Atoh1-CreER knock-in mice show that many cells that initially
express Atoh1 subsequently differentiate into supporting cells rather than hair cells,
suggesting an active competition between neighboring progenitor cells (Driver et al.,
2013; Yang et al., 2010) .Our data showing that while no hair cell differentiation occurs
in the Atoh1 knockout mouse (Bermingham et al., 1999), ATOH1
+
hair cells are clearly
detected in the Pou4f3 mutant organ of Corti in a normal pattern, suggesting that the
Notch-dependent selection process has already occurred before POU4F3 begins to
function in the cochlea, highlighting the stepwise process of differentiation. Thus,
POU4F3 is not involved in hair cell selection among the prosensory cells that express
low levels of ATOH1, and which appear as an equivalence group within the developing
organ of Corti (Abdolazimi et al., 2016; Cai et al., 2013; Tateya et al., 2019), but rather
is required for subsequent ATOH1-dependent survival and differentiation.
Our data clearly demonstrate a need for synergy between ATOH1 and POU4F3 in
positively promoting hair cell differentiation. However, our RNAseq analysis of Pou4f3-
47
null mice also supports a role for these factors in repressing alternative neurosensory
fates. We show that many genes normally expressed by spiral ganglion neurons that
innervate hair cell genes are ectopically expressed in the Pou4f3 mutant hair cells,
including NeuroD1, Pou4f1 and Lhx2 (data not shown), suggesting that the de-
repression of a neuronal differentiation program in Pou4f3 mutants is due to the loss of
an active inhibitory mechanism. Evidence suggests that this active inhibitory mechanism
in wild type hair cells may be mediated by the transcriptional repressor GFI1 (Costa et
al., 2019; Wallis et al., 2003), which we show is also one of the POU4F3-dependent
ATOH1 targets in hair cells (Figure 4G). Together, these data suggest that the
ATOH1/POU4F3 feed-forward synergy may indirectly repress neuronal programs such
as axon formation in order to generate the axon-less mechanosensory hair cells.
The pioneer activity of POU4F3 is necessary for its feed-forward synergy with
ATOH1
Nucleosomes are generally considered a barrier to enhancer activation by preventing
transcription factors binding to DNA (Gaykalova et al., 2015; Petesch and Lis, 2012;
Weber et al., 2014). A select group of transcription factors called pioneer factors
(Iwafuchi-Doi and Zaret, 2014, 2016; Soufi et al., 2015) are able to overcome these
barriers through their ability to recognize potential enhancer regions in silent chromatin.
By allowing access to regions of the genome that are inaccessible in a particular cell
type, pioneer factors play essential roles during development, regeneration and in the
reprogramming of cell fates (Cirillo et al., 2002; Lee et al., 2005; Mayran et al., 2018;
Soufi et al., 2015). Our data show that during inner ear development, POU4F3 is
48
required for chromatin accessibility at
~
45% of de novo ATOH1 targets, as well as a
large number of open chromatin regions in hair cells that are the likely targets of other
classes of hair cell transcription factors (data not shown). We show that POU4F3 binds
to closed chromatin at putative hair cell enhancers in MEFs (Figure 5B), but that ATOH1
is unable to bind these sites in either MEFs or Pou4f3 mutant hair cells (Figure 4C, 5B).
What is the basis for POU4F3’s pioneer activity? The POU family of transcription
factors contain a highly conserved, bipartite DNA binding domain that includes a POU-
specific domain and a POU-homeodomain separated from each other by an
unstructured linker region (Verrijzer et al., 1991, 1992). Both these domains bind to the
major grooves on opposite sides of the DNA double helix (Herr and Cleary, 1995), but
the two domains can bind independently, albeit at a lower affinity (Malik et al., 2018). In
the case of the POU-domain protein OCT4, it has been proposed that these
independent DNA binding domains can render chromatin accessible, first by engaging
the surface of nucleosomes containing half-site motifs to induce nucleosome
“breathing”, and then by allowing the cooperative binding of both domains in open DNA
to achieve a higher affinity (Soufi et al., 2015). Of note, our data in MEFs expressing
POU4F3 and ATOH1 show that the binding of POU4F3 to nucleosomal DNA also
allows ATOH1 to bind to a subset of its target motifs, even in the absence of major
chromatin rearrangements as detected by µATACseq (Figure 5B). However, the
physical mechanism by which POU4F3 allows ATOH1 to access its target motifs in
nucleosomal chromatin remains uncertain.
49
At present, OCT4 and POU4F3 are the only POU-domain transcription factors
demonstrated to have pioneer factor activity, and the ability to bind nucleosomal DNA.
In contrast, the POU3-family transcription factor POU3F2 (BRN2), which can reprogram
mouse embryo fibroblasts to a neuronal state in the context of the BAM cocktail (Brn2,
Ascl1 and Myc1), was shown to have no pioneer factor activity (Vierbuchen et al., 2010;
Wapinski et al., 2013). Instead, the BAM reprogramming cocktail relies on the bHLH
transcription factor ASCL1 to bind nucleosomal DNA and induce chromatin re-
arrangement and subsequent reprogramming. Given the high degree of conservation
between POU transcription factors, it is unclear why some POU factors have pioneer
activity while others do not.
The role of POU4F3 and ATOH1 in the evolution of vertebrate mechanoreceptors
Atoh1 is expressed in developing hair cells and Merkel cells (Bermingham et al., 1999;
Lumpkin et al., 2003; Maricich et al., 2009b), intestinal secretory cell lineages (Yang et
al., 2001), cerebellar granule precursors (Ben-Arie et al., 1997) and subpopulations of
excitatory neurons in the hindbrain (Helms and Johnson, 1998; Rose et al., 2009).
Pou4f3 is expressed in a variety of cell types, such as hair cells (Xiang et al., 1997b,
1998), Merkel cells (Masuda et al., 2011), dorsal root ganglion neurons (Badea et al.,
2012) and some retinal ganglion neurons (Sajgo et al., 2017). However, combinatorial
expression of Atoh1 and Pou4f3 has so far only been observed in hair cells and Merkel
cells, suggesting that the ATOH1/POU4F3 feed-forward synergy that we describe may
have a general role of promoting mechanosensory differentiation (Badea et al., 2012).
Indeed, we were able to identify over 800 POU4F3-dependent ATOH1 targets shared
50
between hair cells and Merkel cells, including a common putative enhancer in the
PIEZO2 gene, which encodes a pore-forming mechanotransduction channel expressed
in hair cells and Merkel cells (Figure 8D). Nevertheless, despite having some
ATOH1/POU4F3 targets in common, hair cells and Merkel cells are morphologically and
functionally very different cell types, and our data clearly show that additional distinct
enhancer networks operate in each cell type (Figure 8A, B). For example, stereocilia
components such as Grxcr1, Lhfpl5, Ush2a, Cdh23, and Myo3b are targets of ATOH1
and POU4F3 in hair cells, but not in Merkel cells (Figure 8D and Figure 9). Conversely,
many POU4F3-dependent ATOH1 targets in Merkel cells group under GO terms such
as integrin signaling and inward-rectifying potassium channels that are not prominently
featured in hair cells (Figure 9). Consistent with our data, cell type-specific accessibility
of binding sites for pleiotropic pioneer factors has also been observed for FOXA2,
GATA4 and OCT4 in other systems (Donaghey et al., 2018).
What are the mechanisms that selectively open and activate the POU4F3-
pioneered, ATOH1-bound distal elements in functionally related, but distinct
mechanosensory cell types? This specificity is not simply a product of POU4F3 binding
affinity: as mentioned earlier, we observed that POU4F3 can weakly bind to
nucleosome-occupied chromatin of some hair cell-specific targets in Merkel cells, and
some Merkel cell-specific targets in hair cells (Figure 8B). We suggest that other cell
type-specific determinants are likely to be required for the deployment of the POU4F3-
pioneered, ATOH1-bound distal elements. When we overexpressed POU4F3 and
ATOH1 in mouse embryo fibroblasts that lack most hair cell or Merkel cell-specific
51
transcription factors, most POU4F3-dependent ATOH1 targets remained closed (Figure
5B, upper panel), even though they are bound by both transcription factors (Figure 5B,
lower panel). Motif enrichment analysis, and our immunostaining results, suggest that
expression of POU4F3-dependent ATOH1 target hair cell genes may be potentiated by
SIX and GATA transcription factor families in hair cells (Figure 8C, and Figure 10), while
expression of POU4F3-dependent ATOH1 target Merkel cell genes may be potentiated
by TFAP2 and SOX family members in Merkel cells (Figure 7A, and Figure 8C).
Interestingly, recent studies show TFAP2 transcription factors are strong nucleosome
binders (Fernandez Garcia et al., 2019), and may exhibit pioneer factor activity during
the differentiation of surface ectoderm progenitors from embryonic stem cells (Li et al.,
2019). These observations provide an opportunity to test the contribution of these cell
type-specific transcription factors in the establishment of cell type-specific gene
expression in two distinct mechanosensory cells - hair cells and Merkel cells.
Are different combinations of transcription factors from the bHLH family and the
POU-IV family responsible for the increasing complexity in the evolution of
mechanosensory organs? In the mechanosensory module of the nematode
Caenorhabditis elegans, the single bHLH and POU-IV homologs Lin-32 and Unc-86 are
both indispensable for the formation of their six touch receptor neurons (Chalfie and Au,
1989). In contrast, in the mouse inner ear, distinct combinations of bHLH and POU-IV
homologs are required for the differentiation of hair cells (Atoh1 and Pou4f3), and the
sensory neurons that innervate them (NeuroD1 and Pou4f1). It is likely that successive
duplication of bHLH and POU family members during chordate evolution permitted the
52
division of labor of a mechanosensitive sensory neuron into two cell types - a
mechanosensitive secondary receptor cell (hair cells or Merkel cells) and a primary
sensory neuron (spiral ganglion neurons of the inner ear, or SA1 afferent neurons of the
skin). It is interesting to speculate on the relationship of these epidermally-derived
Merkel cells versus placodally-derived hair cells to the primary and secondary sensory
receptor cells seen in non-vertebrate chordates, such as the oral siphon hair cell-like
cells of urochordates and the epidermal sensory neurons of cephalochordates (Burighel
et al., 2008, 2011; Gasparini et al., 2013), and whether the POU4F3/ATOH1 feed-
forward mechanism we describe here also plays a role in the development of
mechanosensory cells throughout the chordate lineage.
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Chapter 3. Enhancer decommissioning during inner ear
maturation imposes an epigenetic barrier to sensory hair cell
regeneration.
INTRODUCTION
The inability to regenerate damaged tissues, such as heart, brain, retina, kidney, and
the inner ear, is a major cause of human disability (Atkinson et al., 2015; Goldman,
2014; Hashimoto et al., 2018; Humphreys, 2014; Martino et al., 2011; Mittal et al.,
2017). Although they appear to lack tissue-resident stem cells, several of these tissues
show a limited degree of regenerative potential at perinatal stages in mice, but full
regenerative capacity in other species.
In non-mammalian vertebrates, loss of hair cells can stimulate a robust regenerative
response among the surviving supporting cells through two different mechanisms: (1)
cell cycle re-entry followed by re-differentiation into both hair cells and supporting cells;
(2) direct transdifferentiation into hair cells without cell proliferation (Brignull et al., 2009;
Li et al., 2016). While in the mammalian organ of Corti, the sensory organ of the
cochlea, hair cell damage can lead to permanent deafness since hair cells do not
naturally regenerate in adults (White et al., 2006). We and others have identified a short
postnatal time window, in which supporting cells in the mouse organ of Corti can be
forced to transdifferentiate into hair cells by pharmacologically blocking Notch signaling
(Doetzlhofer et al., 2009; Stojanova et al., 2016). However, this transdifferentiation
potential is lost by postnatal day 6 (P6). These observations suggest that the
regenerative capacity of postnatal supporting cells may be still latent, but that specific
54
mechanisms associated with maturation may be responsible for the failure of their
transdifferentiation in adults.
In Chapter 3, we investigated the chromatin structure and the epigenetic changes
accompanying the perinatal development of cochlear supporting cells in mice, focusing
our analysis on the genes that are specifically expressed in hair cells identified by
RNAseq, and their potential regulatory elements, revealed by ATACseq and CUT&RUN
techniques. We discovered that the hair cell gene regulatory network is maintained in a
transcriptionally silent state in nascent supporting cells. This is evident by (1) the
presence of repressive histone modification H3K27me3 at the hair cell-specific gene
promoters, (2) the presence of “priming” histone modification H3K4me1 at the hair cell-
specific gene enhancers, (3) and the lacking of active histone modification H3K27ac at
the hair cell-specific gene promoters and enhancers, in nascent supporting cells. This
silent epigenetic state of hair cell gene regulatory network in nascent supporting cells
could be reversed upon Notch inhibition, but only at perinatal stages. We found that the
loss of supporting cell transdifferentiation potential during their postnatal maturation
correlates with their gradual loss of H3K4me1 at the hair cell-specific gene enhancers.
Inhibition of the DNA methyltransferase that removes H3K4me1 in cultured cochlear
supporting cells partially preserved their transdifferentiation potential.
55
RESUTLS
Active enhancers around the hair cell-specific genes are mostly ATOH1 targets
To identify the genes and the associated regulatory elements that separate the hair cell
and supporting cell fates during the development of the organ of Corti, we first
compared the transcriptome of FACS-purified postnatal day 1 (P1) hair cells and P1
supporting cells, using Atoh1-GFP and Lfng-GFP transgenic mouse lines, respectively
(Figure 11A). We found that 949 genes were expressed in the hair cells at significantly
higher levels (Adjusted p value < 0.01; fold change > 2, FPKM in hair cells > 1; DESeq2
baseMean > 50) (Figure 11A), including many known hair cell markers, such as Atoh1,
Pou4f3, Gfi1, Myo6, Jag2, etc. These hair cell-specific genes are enriched for cilium
assembly and sensory perception of sound as gene ontology (GO) terms (Figure 11A).
We next sought to identify the regulatory elements for the hair cell-specific genes
that we have defined. Although genes with multiple isoforms may be present in the hair
cells, we define the proximal regulatory element (promoter) of each gene as the
genomic region around the transcription start site (TSS) of its most abundant transcript
in our RNAseq data (data not shown). To identify the potential distal regulatory
elements for each hair cell-specific genes, we profiled the chromatin accessibility of hair
cells using μATACseq (Figure S1, Buenrostro et al., 2013), and histone modifications
associated with active enhancers (H3K4me1 and H3K27ac) using CUT&RUN (Skene
and Henikoff, 2017). We focused our analysis on the 14,243 accessible distal elements
that are within 200 kb around the 949 hair cell-specific genes. Base on their levels of
H3K4me1 and H3K27ac, we classified them into four groups (Figure 11B). For example,
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Figure 11. Analysis of the hair cell epigenome defines a class of “active” putative hair cell enhancers at
postnatal day 1, as well as the ATOH1 hair cell targetome. (A) Transcriptomic analysis of FACS-purified P1 hair
cells and supporting cells. The organ of Corti matures within the cochlear duct in a basal-to-apical wave as indicated,
and in cross-section consists of one row of inner (IHC) and three rows of outer (OHC) hair cells (expressing an
Atoh1-GFP transgene), as well as a variety of supporting cells types that express a Lunatic fringe-GFP transgene
(Lfng-GFP transgenes - labeled red with green outline). Postnatal day 1 organs were dissociated, FACS-purified, and
analyzed by RNAseq (see Methods) for hair cell- and supporting cell-enriched genes. Genes with fold change >2
(log2(fold change) >1) and p value < 0.01 (-log10(p value) >2) are defined as hair cell genes (green dots), and genes
with fold change < 0.5 (log2(fold change) < -2) and p value < 0.01 (-log10(p value) >2) are defined as supporting cell
genes (red dots). Gene ontology analysis against Biological Process database indicates terms derived from the hair
cell-enriched gene set using EnrichR (Chen et al., 2013). (B) Categorical heatmaps showing putative hair cell-gene
enhancer prediction based on chromatin accessibility, H3K4me1 and H3K27ac. Genomic regions depicting potential
hair cell gene-distal regulatory elements were defined by “open” chromatin structure (ATACseq peaks, ±5kb for
heatmap), lying within ±200kb of hair cell-enriched gene TSS. Regions ±2kb from TSS were excluded, and rows were
ranked by their genomic coordinate within each cluster. There are 2894 putative “active” enhancers enriched for both
H3K4me1 and H3K27ac, 4239 “primed” enhancers marked by H3K4me1 alone; 3407 “unmarked” elements and 256
possible “unannotated” promoters enriched for H3K27ac only. ATOH1 CUT&RUN signals indicated that a high
proportion of active enhancers are targets of ATOH1 binding. FACS-purified, Atoh1-GFP+ hair cells were used (2-3
replicates). (C) Representative IGV gene tracks of hair cell-enriched genes Atoh1 and Rasd2 showing putative
enhancers (grey bars) identified by epigenetic characteristics as indicated (B). Known Atoh1 3’ enhancer is
accessible (ATAC peak), H3K4me1- and H3K27ac-enriched, and bound by ATOH1. Predicted Rasd2 5’ enhancer is
similarly labled by H3K4me1 and H3K27ac. (D) De novo generated ATOH1-binding motif from E17.5 hair cells (the
time of peak Atoh1 cochlear expression (Stojanova et al, 2015)). Top 2,000 ATOH1-bound peaks were used for motif
enrichement analysis by HOMER (Heinz et al., 2010). Motif shown was highly enriched with p value of 1e-399 and
found in 71% of top 2,000 peaks.
57
a very small portion of these accessible regions, which we thought were distal, was
labeled with high levels of H3K27ac, but not H3K4me1 (Figure 11B, group three),
suggesting that these regions are likely to be unannotated promoters (Creyghton et al.,
2010), thus are excluded from further analysis.
In particular, we are interested in the 3,970 distal regulatory elements (Figure 11B,
group one), which include the known 3’ autoregulatory element for Atoh1 expression
(Figure 11C), that are highly enriched for H3K27ac and H3K4me1, as they may function
as active enhancers (Calo and Wysocka, 2013; Creyghton et al., 2010). Indeed, these
regions are remarkably enriched for the binding of ATOH1 (Figure 11B, C). In contrast,
the 5,483 accessible regions, which are only enriched for H3K4me1 but not H3K27ac
(Figure 11B, group 2), have very low levels of ATOH1 signal in hair cells. These data
suggest that the binding of ATOH1 is likely to cause the transition from the primed to the
active enhancer during hair cell differentiation, by recruiting mechanisms to add
H3K27ac to the distal regulatory elements. Interestingly, motif enrichment analysis of
the top 2,000 ATOH1 binding sites revealed a 10 bp motif (Figure 1D) that is
significantly different from the ATOH1 motif previously identified in the cerebellar
granule precursors (Klisch et al., 2011), another Atoh1-expressing cell type, indicating
the binding of ATOH1 to specific open distal elements is likely to be influenced by the
presence of co-factors. Consistent with these observations, the 5,483 primed distal
regulatory elements are also enriched for E-box motifs (data not shown), even though
the level of ATOH1 binding at these regions is very low (Figure 11B). We confirmed that
our analysis is not biased to hair cells, since we also identified 1,251 supporting cell-
58
specific genes, and 3,227 distal regulatory elements around these genes (data not
shown), which are labeled with high levels of H3K4me1 and H3K27ac, including the
ones near Hes5, Id2, Jag1, etc. Together, these data suggest that both hair cell and
supporting cell fates are actively driven by their specific gene regulatory networks
(active enhancers and transcription factors).
Hair cell gene promoters are poised in the perinatal supporting cells
The latent potential for supporting cells to transdifferentiate into hair cell during the first
postnatal weeks of mouse development suggests that the hair cell-specific gene
regulatory program may be temporarily available in the perinatal supporting cells.
Previous studies from our lab showed that the transcription of Atoh1 is repressed in the
nascent supporting cells, by active de-acetylation at the promoter region mediated via
Notch signaling effectors HES/HEY transcription repressors (Abdolazimi et al., 2016). In
addition, our lab also found that the promoter of Atoh1 is marked by both histone
modifications H3K27me3 and H3K4me3 in the perinatal supporting cells, using ChIP-
qPCR (Stojanova et al., 2016). The combinatory presence of both the repressive
(H3K27me3) and permissive (H3K4me3) epigenetic markers suggests that Atoh1 is
poised at a bivalent state. We hypothesize that the same epigenetic mechanisms also
apply to the other hair cell-specific genes to render their low expression levels in the
perinatal supporting cells.
To test this hypothesis, we compared the levels of chromatin accessibility and
epigenetic marks at the hair cell-specific gene promoters between P1 hair cells and P1
supporting cells. The 949 hair cell-specific promoters are all open and labeled by the
59
H3K4me3 in both cell types (Figure 12A), a histone modification associated with
positive regulation of transcription around TSS, indicating that these promoters are
accessible to the binding of transcription machineries to allow gene expression. As
expected, these promoters are also marked by significant lower levels of the active
histone marker H3K27ac in P1 supporting cells, and significantly higher levels of the
repressive signal H3K27me3 (Figure 12A), compared to those in P1 hair cells,
consistent with the low expression levels of hair cell-specific genes in the supporting
cells (Figure 11A). Together, these results suggest that, as previously shown for Atoh1
locus, the promoters of hair cell-specific genes are maintained in a poised state, which
is typically observed around the developmental genes in the embryonic stem cells
(Bernstein et al., 2006).
Hair cell-specific enhancers are also poised in the perinatal supporting cells
As we have shown in Chapter 2, during the embryonic development of the cochlea, in
the undifferentiated progenitors that contain future hair cells and supporting cells,
ATOH1 initially binds to the distal regulatory elements pre-established (open and
labeled by H3K4me1) prior to the upregulation of Atoh1 (Figure 1A). Genes associated
with these pre-established ATOH1 targets include Notch signaling ligands Jag2 and
Dll1, and transcription factors Pou4f3, etc. (Figure 1E and Figure 6C), which allow
ATOH1 to rapidly initiates lateral inhibition, and further hair cell differentiation.
Interestingly, we found that the hair cell-specific distal regulatory elements that we
identified from P1 hair cells, which are mostly ATOH1 targets (Figure 11B), are also
open and marked by H3K4me1 in the P1 supporting cells. In addition, we found that
60
Figure 12. The hair cell gene regulatory network is “primed”, but silenced by both H3K27me3 and active
HDAC activity in P1 supporting cells. (A) Chromatin structure (ATACseq) and epigenetic analysis (H3K4me3;
H3K4me1, H3K27ac, and H3K27me3) were conducted on FACS-purified P1 hair cells (P1HC, Atoh1-GFP+) and
supporting cells (P1SC, Lfng-GFP+) to determine the disposition of HC-gene regulatory elements. Heat maps for both
Promoter regions (±2kb TSS) and putative enhancers (defined in Figure 1B) are shown, along with associated
average signal intensity profiles for P1HC and P1SC. HC-gene promoters are accessible (ATACseq) in bothP1HCs
and P1SCs, as well as being marked by H3K4me3, and devoid of H3K4me1. HC-gene promoters show increased
H3K27ac in HCs over SCs, and show significantly increased H3K27me3 silencing in SCs over their counterparts in
HCs. HC-gene enhancer elements are also accessible in both P1HCs and P1SCs, as well as being primed by
H3K4me1. HC-gene enhancers show lower levels of H3K27ac activation and increased levels of H3K27me3 in SCs
relative to the same elements in HCs. (B) Quantification of primed (H3K4me1/3+ and H3K27ac-) hair cell gene
promoters and enhancers in P1HC and P1SC. Approximately 52% of HC-gene promoters (498 promoters) are
primed in supporting cells, while less than 7% (72 promoters) are classified as primed in hair cells. Forty percent of
HC-gene enhancers (1,159 enhancers) are primed in supporting cells, while only 3% (86 enhancers) are classified as
primed in hair cells. (C) IGV gene track views of hair cell genes Atoh1 and Rasd2, illustrating the differential
epigenetic state in P1HC and P1SC. The Atoh1 promoter is accessible and marked by H3K4me3 in both cell types,
but is enriched for H3K27ac only in P1HCs. The Atoh1 3’ enhancer (grey box) is accessible and enriched for
H3K4me1 in both cell types, but it lacks H3K27ac in P1SC. Similarly, the Rasd2 promoter is marked by H3K4me3
and the Rasd2 enhancer (grey box) is marked by H3K4me1 in both P1HC and P1SC, but both elements are
differentially marked by H3K27ac and H3K27me3. (D) Inhibition of H3K27 deacetylase (TSA) leads to up-regulation
of HC-gene expression in FACS-purified supporting cells. Organ of Corti explants were treated with either control
(DMSO) or Histone deacetylase (HDAC) inhibitor (TSA; 200nM, 48hr) followed by dissociation, FACS-purification of
supporting cells (Lfng-GFP+) and RNAseq analysis. (E) Inhibition of H3K27 methyltransferase (GSK343) leads to up-
regulation of HC-gene expression in FACS-purified supporting cells. Organ of Corti explants were treated with either
control (DMSO) or histone methyltransferase inhibitor (EZH2 inhibitor GSK-343, 5µM, 48hr). Error bars stand for data
range, *** means adjusted p value < 0.001, ** means adjusted p value < 0.01 and * means adjusted p value < 0.05.
61
these primed ATOH1 targets have significantly lower levels of H3K27ac, and higher
levels of the repressive marker H3K27me3 in P1 supporting cells, compared to those in
P1 hair cells. These observations suggest that the activity of hair cell-specific enhancers
may also be actively de-activated / repressed in the supporting cells.
Taken together, our data indicate that the epigenetic states of hair cell-specific
genes in perinatal supporting cells, namely the poised promoters and the poised
enhancers, are likely to be involved in the maintenance of the latent potential for their
transdifferentiation upon hair cell damage.
Low expression of hair cell-specific genes in supporting cells is maintained by
active histone de-acetylation and histone methylation
Next we tested whether the maintenance of the de-acetylated and methylated states at
H3K27 are important for silencing the hair cell gene regulatory program in the perinatal
supporting cells. To do this, we investigated whether perturbation of the epigenetic
mechanisms described above could de-repress the expression of hair cell-specific
genes in perinatal supporting cells.
We dissected P1 cochlea from Lfng-GFP mice, and cultured them in vitro for 48h
with the presence of histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) to block
histone de-acetylation (Kim and Bae, 2011; Vigushin et al., 2001); or with histone
methyltransferase EZH2 inhibitor GSK-343 to block the addition of H3K27me3 (Yu et
al., 2017; Zhou et al., 2019). Subsequently, we FACS-purified the supporting cells, and
used RNAseq to profile their gene expression after inhibitor treatment. Similar sets of
hair cell-specific genes were significantly upregulated in either TSA treated or GSK-343
62
treated supporting cells (Figure 12D, E), including Atoh1, Pou4f3, Gfi1 and Rasd2, etc.,
indicating that these two mechanisms are co-regulating the expression of hair cell
genes in the supporting cells. TSA treatment or GSK-343 treatment also decreased the
expression of supporting cells genes (data not shown), suggesting a tendency for their
cell fate transition. However, neither TSA treatment alone or GSK-343 alone was able to
fully induce the transdifferentiation of supporting cells into hair cells, as determined by
MYO6 immunostaining, suggesting that histone de-acetylation may work independently
from the histone methylation at the hair cell-specific gene loci in perinatal supporting
cells.
Separating responding and non-responding supporting cells from perinatal
cochlea during transdifferentiation
After hair cell damage, supporting cells in non-mammalian vertebrate sensory epithelia
are able to direct transdifferentiate into hair cells (Brignull et al., 2009). This direct
transdifferentiation is partially initiated by the lack of Notch-mediated lateral inhibition,
due to the adjacent hair cell loss. Similarly, in the in vitro cultured perinatal mouse
cochlea, supporting cell transdifferentiation can be achieved by Notch inhibition using
gamma secretase inhibitor DAPT (Doetzlhofer et al., 2009). However, the
responsiveness of supporting cells to Notch inhibition is diminished after the first
postnatal week during development, correlating with their loss of transdifferentiation
potential upon hair cell damage. Since transdifferentiating and non-transdifferentiating
supporting cells co-exist in the DAPT treated cochlear explants at perinatal stages, we
63
reasoned that this gives us an opportunity to reveal the barriers for transdifferentiation,
by investigating and comparing these two populations of supporting cells.
To do this, we in vitro cultured cochleae dissected from Lfng-CreER; Rosa-
tdTomato; Atoh1-GFP mice at P1, in which supporting cells were permanently labeled
with tdTomato expression by adding tamoxifen. After DAPT treatment, the
transdifferentiating supporting cells (responding) were recognized by their ATOH1-GFP
upregulation, while the non-transdifferentiating supporting cells (non-responsding)
remained single-positive for tdTomato expression (Figure 13A). We quantified the ratio
of responding versus non-responding supporting cells, by analyzing the dissociated
organ after culture using FACS. As expected, as the supporting cells mature, the ratio of
responding cells decreased dramatically (Figure 14A, B). At E17.5, most of the labeled
supporting cells are able to upregulate ATOH1-GFP to the level comparison to those in
hair cells. In contrast, at P6, only 1.9% of the labeled supporting cells were able to
upregulate ATOH1-GFP after DAPT treatment (Figure 14B).
Supporting cell transdifferentiation is accompanied by activation of the poised
hair cell-specific enhancers, and the acetylation of the poised promoters
To investigate whether the ATOH1-GFP positive responding cells also express the
other hair cell-specific genes, we profiled the transcriptome of the FACS-purified
double-labeled cells from DAPT treated P1 culture, which is the time point when
approximate half of the supporting cells were responding (Figure 13A and Figure 14B).
As control, we profiled the transcriptome of the FACS-purified tdTomato-positive
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Figure 13. Transdifferentiation of supporting cells is accompanied by HC-gene enhancer activation
(H3K27ac) and increased chromatin accessibility (ATACseq). (A) RNAseq analysis of transdifferentiation.
Schematic showing the strategy for purifying subpopulations of supporting cells based on their transdifferentiation
potential (response to Notch inhibition). P1 organ of Corti explants from transgenic mice harboring both the Lfng-
CreERT2/tdTomato reporter ( Semerci et al., 2017) to lineage-trace supporting cells, as well as the the Atoh11-GFP
transgene were treated with tamoxifen (24hr) to label existing supporting cells (red), while hair cells express the
Atoh1-GFP transgene (green). Organs are then treated with control (DMSO) or Notch inhibitor (DAPT - 10µM).
Supporting cells undergoing transdifferentiation express both transgenes (seen as yellow in FACS plot – Permissive),
while supporting cells that are refractory to loss of Notch signaling (Non-permissive) remain red-only. Supporting cells
from control organs (pink box), Permissive (yellow box) and Non-permissive (red box) were also collected and used
to construct libraries for RNAseq. Scatter plots showing gene expression changes in Non-permissive and Permissive
subpopulations of supporting cells relative to control. Hair cell genes with significant expression changes (fold change
>2 or < -2 and p value < 0.01) are colored in green, and supporting cell genes are colored in red. Atoh1, Pou4f3,
Lhx3, and Gfi1 are indicated as examples of hair cell genes that are strongly induced during transdifferentiation. (B)
Chromatin structure (ATAC) and epigenetic change (H3K27ac) accompanying transdifferentiation was compared at
HC-gene promoters and enhancers in Control, Non-permissive, and Permissive (transdifferentiating) supporting cells
populations. Comparison of heat-maps and average signal profiles for each group, indicates that HC-gene
enhancers gain both accessibility and H3K27ac levels during transdifferentiation (compare Permissive with Non-
permissive and Control), while promoters are already accessible, and gain modest H3K27ac levels during
transdifferentiation. (C) Examples at the gene level of changing epigenetic status upon transdifferentiation (IGV gene
tracks) for hair cell-genes Atoh1 and Pou4f3. Both show epigenetic activation (increase in H3K27ac) in supporting
cells during transdifferentiation. Promoters and enhancers (grey boxes) of those genes gain accessibility (higher
ATAC peaks), and accumulate H3K27ac levels in Permissive supporting cells.
65
supporting cells after treatment with DMSO, the solvent for DAPT. Among the 949 hair
cell-specific genes, ~2/3 were highly upregulated in the permissive supporting cells,
including Atoh1, Pou4f3, Gfi1, Lhx3, etc. (Figure 13A), suggesting that the hair cell
program was fully activated during the transdifferentiation of these permissive cells.
Consistent with the upregulation of most of the hair cell-specific genes, we found
that the hair cell-specific promoters have significantly higher levels of the active histone
marker H3K27ac in the responding supporting cells compared to those in the control
(Figure 13B, C). In addition, both the accessibility and the level of H3K27ac increased
dramatically at the hair cell-specific enhancers in the responding supporting cells.
Hair cell-specific gene promoters are kept bivalent during supporting cell
maturation
In contrast, around 53% of the labeled supporting cells at P1 were not ATOH1-GFP
positive after DAPT treatment (Figure 13A and Figure 14B). This could be due to the
fact that the Notch signaling components decrease their expression during the first
postnatal week of cochlear development, as previously shown, rendering the supporting
cells insensitive to DAPT. However, RNAseq data showed that the expression of Hes5
were among the most significantly downregulated genes in the non-responding cells as
a result of Notch inhibition (Figure 13A). In addition, the expression of a few hair cell-
specific genes, including Atoh1 and Pou4f3, were also significantly upregulated in the
non-responding cells (Figure 13A), although to a much lesser extent compared to those
in the responding cells after DAPT treatment. These data indicate that the inability for
66
transdifferentiation in the non-responding supporting cells was limited by mechanisms
different from insensitive to Notch inhibition.
During the differentiation of embryonic stem cells, resolution of the poised epigenetic
states at the developmental genes correlates with their changes of expression
(Bernstein et al., 2006). To be specific, it was found that co-existence of H3K4me3 and
H3K27me3 at the promoter regions are associated with low levels of gene expression.
Loss of H3K4me3 at the promoters correlates with gene silencing, possibly due to the
loss of the ability to recruit enzyme for nucleosome remodeling and histone acetylation.
On the other hand, loss of H3K27me3 is associated with gene activation. In order to
know whether inability to transdifferentiate of the non-responding supporting cells is a
result of the resolution of the bivalency to a repressive state in those supporting cells,
we profiled and compared the level of H3K4me3 in supporting cells at different
developmental stages.
Surprisingly, we found that the levels of H3K4me3 at the hair cell-specific promoters
remain unchanged during the perinatal and postnatal development of the supporting
cells (Figure 14C), even though the there was a dramatically increase of non-
responding cells from E17.5 to P6 (Figure 14A, B). Together, these data show that the
hair cell-specific gene promoters are kept poised in the maturing supporting cells, and
that the loss of transdifferentiation potential is not likely to be due to the change of the
epigenetic states at the promoters.
Decommission of the hair cell-specific enhancer correlates with loss of
transdifferentiation potential in supporting cells
67
Figure 14. Decommissioning of HC-gene enhancers (H3K4me1) accompanies postnatal maturation of
supporting cells. (A) Organ of Corti explants from P1 and P6 cochlea (surface preparations from Atoh1-Gfp
transgenic mice showing three rows of outer hair cells (bracket) and one row of inner hair cells (arrow)), were
maintained in culture for 72hr and either remained untreated (DMSO control), or treated (DAPT,10µM) to induce
transdifferentiation. Notch inhibition induces ectopic hair cells in P1 organs, but not in P6 organs. Scale bar
represents 50µm. (B) Progressive loss of transdifferentiation potential within the supporting cell population from E17.5
to P6. Organ explants, starting at several developmental stages (E17.5, P1, P3, P6), were cultured (as in Figure 3A)
and treate with DAPT for 48hr; ,and transdifferentiation-permissive supporting cells were quantified by FACS. The
percentage of permissive supporting cells was calculated by dividing GFP+/tdTomato+ cells by total tdTomato+ cells.
The percentage of permissive supporting cells declines as the organs mature (63.9% in E17.5 organs; 47.0% in P1;
31.9% in P3; and 1.9% in P6). Error bars: standard deviations; ***p value < 0.001, n>=3. (C) Promoters of HC-genes
in supporting cells remain unchanged between E17.5 and P6 with regard to H3K4me3 promoter-priming. Heat-maps
and average signal profiles showing similar epigenetic profiles surrounding hair cell gene promoters during supporting
cell perinatal maturation. (D) Enhancers of HC-genes in supporting cells show strongly diminished H3K4me1
enhancer priming (heat-maps and average signal profiles) between E17.5 and P6 during supporting cell perinatal
maturation. (E) IGV gene tracks showing enhancers (grey boxes) associated with Atoh1, Gfi1 and Dll1 are depleted
of H3K4me1 in P6 supporting cells, but H3K4me3 is maintained at their promoters. Y-axis normalized within each
sample type (ATAC, H3K4me3 and H3K4me1).
68
During development, upregulation of Atoh1 to generate sufficient ATOH1 protein
involves the activation of its 3’ autoregulatory enhancer, as a key element in its feed-
forward regulatory loop. The observation that Atoh1 transcription was significantly but
not as dramatically increased in the non-responding cells compared to the responding
supporting cells suggests that this 3’ autoregulatory enhancer was not activated in the
non-responding cells upon DAPT treatment. Consistent with this observation, we found
that this Atoh1 3’ autoregulatory enhancer was barely accessible, and was not labeled
with H3K27ac in the non-responding supporting cells after Notch inhibition (Figure 14E).
Similar inactive epigenetic states were also found around the other hair cell-specific
enhancers in the non-responding supporting cells after DAPT treatment (Figure 14E).
Therefore we hypothesize that the epigenetic changes in the non-responding cells at
the hair cell-specific enhancers, which are mostly ATOH1 targets, limits the accessibility
of ATOH1 to those regulatory element during Notch inhibition, and thus causes the
failure of their transdifferentiation.
Interestingly, among the epigenetic markers that we have profiled, we found that
the levels of H3K4me1 decreased dramatically in P6 supporting cells compared to those
in P1 supporting cells at the hair cell-specific enhancers (Figure 14D), temporarily
matching the dramatic loss of transdifferentiation potential of the maturing supporting
cells. In addition, we found that supporting cells purified from the basal half of the P1
cochleae have significantly lower H3K4me1 levels around the hair cell-specific
enhancers, compared to those in the supporting cells purified from the less matured
apical half (Figure 15A, B). This suggests that the loss of H3K4me1 at the hair cell-
69
specific enhancers propagates in a basal-to-apical wave in the maturing supporting
cells, which is consistent with the basal-to-apical gradient of the loss of
transdifferentiation potential during supporting cell maturation. Taken together, these
data suggest that the loss of transdifferentiation potential from E17.5 to P6 supporting
cells is caused by the loss of H3K4me1 at the hair cell specific enhancers.
To test this, we first clustered the hair cell-gene enhancers into groups that were
H3K4me1-positive, or H3K4me1-negative in P1 supporting cells, and correlated the
DAPT-induced changes in RNA expression of the genes associated with each group of
enhancers (Figure 16A). The genes associated with H3K4me1-positive enhancers were
significantly up-regulated following 24hr of DAPT treatment, compared to those genes
that were associated with enhancers that were H3K4me1-negative (p<0.001; Figure
16A). This correlation between H3K4me1 priming and DAPT-induced transcriptional
induction strongly supports the essential role of hair cell gene enhancer commissioning
by H3K4me1 in determining the transcriptional competence of hair cell genes in
supporting cells.
Second, we reasoned that, if removing of H3K4me1 is responsible for the failure of
transdifferentiation, blocking demethylation of H3K4me1 in the cultured organ of Corti
using the KDM1A inhibitor (GSK-LSD1; Mohammad et al., 2015) would delay or prevent
the loss of supporting cell transdifferentiation observed in Figure 14B. To test this, P1
organ of Corti explants from either the apex or base were pre-incubated with either
control (DMSO) or GSK-LSD1 inhibitor for 24 hours, and treated with DAPT for another
48 hours to induce transdifferentiation. We found that GSK-LSD1 treatment significantly
70
increased the number of responding supporting cells from the basal half of the organ of
Corti (Figure 16B). While no change in the proportion of responding supporting cells
was observed in the apical half (Figure 5B). When whole organ of Corti explants from
P1 or P3 cochlea were tested, an increase in the ratio of responding supporting cells
was observed in P1 culture, but not P3. These results suggest inhibition of the removal
of H3K4me1 can preserve the transdifferentiation potential of the maturing supporting
cells. However, once H3K4me1 is lost at the hair cell-specific gene enhancers, GDK-
LSD1 treatment cannot make a difference.

Figure 15. The “primed” state of hair cell-gene
enhancers (H3K4me1) in supporting cells
determines the transcriptional response during
transdifferentiation (Notch inhibition). (A) Primed
enhancers preferentially drive hair cell gene
expression during P1 supporting cell
transdifferentiation. RNA expression was measured
among genes associated with “primed” enhancers
(H3K4me1+ in P1SC), and compared to genes
associated with “decommissioned” enhancers
(H3K4me1- in P1SC). Comparison of quantile
distribution of RNAseq (log2(Fold change))
following Notch inhibition (DAPT - 10nM – 24hrs).
Error bars represent data range; boxes represent
the first and third quartiles; horizontal line indicates
the median ; Mann-Whitney U test; *** indicates p
value < 0.001. (B) Organ of Corti explants from P1
Atoh1-Gfp and Lfng-CreERT2/tdTomato transgenic
mice (as in Figure 3) were divided into basal and
apical halves, cultured andeither pre-incubated with
GSK-LSD1 (20 µM) for 24 hours, or not pre-treated
as controls. After 24hr pretreatment, the organs were treated either with DAPT (10µM) to induce transdifferentiation,
or DAPT (10µM) + GSK-LSD1 (20 µM) for another 48 hours. Finally, organs were dissociated, and
transdifferentiation-permissive supporting cells were quantified by FACS analysis. The percentage of permissive cells
was calculated by dividing Atoh1-GFP+ and Lfng-CreER/tdTomato+ (double-positive) cells, by total Lfng-
CreER/tdTomato+ cells. A significant increase in the number of permissive supporting cells is seen in the basal half
of the P1 cochlea in the presence of GSK-LSD1 inhibitor, relative to DAPT control (~8% transdifferentiation-
permissive supporting cells by DAPT + GSK-LSD1 vs ~1% by DAPT alone). No significant change is observed in the
apical half of P1 cochlea (~41% in DAPT + GSK-LSD1 and ~42% in DAPT alone). Error bars represent standard
deviations, *** means p value < 0.001 by Student t test, and n>=4. (C) An increase in the percentage of
transdifferentiation-permissive supporting cells is seen at both P1 and P3 following inhibition of LSD1 for 72hrs. The
same experimental paradigm was used as in B, except whole organs of Corti from P1 vs P3 were compared (P1
organs: 32% permissive with DAPT-alone vs 42% by DAPT + GSK-LSD1); (P3 organs: 20% permissive by DAPT-
alone vs 29% by DAPT + GSK-LSD1). Error bars represent standard deviations, ** means p value < 0.01 by student t
test, and n >=8.
71
Discussion
In this Chapter, we investigated the epigenetic mechanisms that control the
transdifferentiation of perinatal cochlear supporting cells into hair cells, upon DAPT-
mediated Notch signaling inhibition. Using CUT&RUN and ATACseq techniques, we
investigated the changes of histone modifications H3K4me1, H3K4me3, H3k27ac,
H3K27me3, and chromatin accessibility during the postnatal maturation of cochlear
supporting cells. We focused our analysis on the hair cell-specific gene loci that we
identified using bioinformatics. We also used a novel in vitro model, in which Notch-
inhibition-responding supporting cells (the upregulation of ATOH1-GFP expression at
the protein level can be detected by FACS) can be separated from non-responding
supporting cells. By comparing these two supporting cell populations at isolated from
cultured P1 cochleae, we found the loss of transdifferentiation potential of the maturing
supporting cells correlated with their the decreasing H3K4me1 level at the hair cell-
specific gene enhancers. Importantly, inhibition of LSD1 (H3K4 methyl-transferease)
preserved the transdifferentiation potential of the supporting cells, suggesting that loss
of H3K4me1 is partially responsible for the loss of transdifferentiation potential of the
maturing supporting cells.
ATOH1 is the master regulator for hair cell specification and differentiation.
Therefore, we use ATOH1-GFP as the reporter in the in vitro model to indicate whether
supporting cells respond to DAPT treatment. Using immunostaining, we found that all of
the ATOH1-GFP+ transdifferentiating supporting cells after DAPT treatment were also
POU4F3+ and MYO7A+ (data not shown). These observations suggest that (1) these
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transdifferentiating supporting cells were turning into hair cells, and (2) ATOH1 is able to
initiate the hair cell gene regulatory network in these supporting cells. Since in
Chapter2, our data suggest that ATOH1 binds to the pre-established and the de novo
targets, and initiates the expression of the associated gene in a hierarchical manner, the
ability of ATOH1 to stimulate the expression of POU4F3 in the transdifferentiating
supporting cells suggest that the pre-established ATOH1 targets are still available in the
responding supporting cells. While, these ATOH1 targets may not be accessible to
ATOH1 in the non-responding supporting cells, which may be the reason why they
could not transdifferentiate upon DAPT treatment.
One of the pre-established ATOH1 targets is the 3’ autoregulatory enhancer for
ATOH1. The current model of Atoh1 upregulation during embryonic development, as
discussed in Chapter 2, has two steps. First, Atoh1 is weakly upregulated in a group of
progenitors. Second, very low levels of ATOH1 protein can activate its 3’ autoregulatory
enhancer to further upregulate Atoh1 expression, in order for full-blown hair cell
differentiation. In the non-responding supporting cells, we did observed the significant
but weak upregulation of Atoh1 mRNA after 48h DAPT treatment, reminiscent to the low
level of Atoh1 in the sensory progenitor cells prior to their differentiation. This analysis
suggests Atoh1 expression is still repressed in those non-responding supporting cells,
and that the failure of their transdifferentiation is not due to the failure of the initial Atoh1
upregulation after Notch inhibition. Although Atoh1 is upregulated, it expression cannot
be further increased, as shown by the FACS analysis of their low ATOH1 GFP level.
73
This result suggests that in the non-responding supporting cells, ATOH1 cannot get
access to its 3’ autoregulatory enhancer.
Interestingly, we found that among the epigenetic markers that we have profiled, the
level of H3K4me1 decreases at the hair cell-specific gene enhancers, including the
Atoh1 3’ autoregulatory enhancer during supporting cell maturation, suggesting the
removal of H3K4me1 limits the access of ATOH1 to its targets. Inhibition of LSD1
increased the proportion of responding supporting cells in the P1 culture, suggesting
that preservation of H3K4me1 maintained the ability of ATOH1 to stimulate the hair cell
program in cultured supporting cells. However, we found that LSD1 inhibition had no
significant effect on the supporting cells from P3 or older cochlea. This is consistent with
the data that H3K4me1 at the hair cell-specific gene enhancers has been already lost in
most of the supporting cells at P3. Our findings are also supported by studies that have
shown that overexpression of ATOH1 in the mature organ of Corti was not sufficient to
induce the transdifferentiation of mature supporting cells to hair cells. However, we do
not understand why/ how the ATOH1 targets get their H3K4me1 removed during
supporting cell maturation. Since we proposed in Chapter2 that de novo ATOH1 targets
can be induced by expression of POU4F3 or other transcription factors. It is likely that
overexpression of ATOH1 and other hair cell specific transcription factors could
transdifferentiate mature supporting cells more efficiently. Consistently, Louise et al., in
our lab have found that overexpression of SIX1, ATOH1, POU4F3 and GFI1 can very
efficiently reprogram both MEFs and mature supporting cells into hair cells. It is not
clear whether overexpression of this battery of transcription factors add h3K4me1 back
74
to the hair cell specific gene enhancers, which enable the access of ATOH1 to its
targets. Since POU4F3 and SIX1 motifs are highly enriched in the de novo ATOH1
targets in hair cells, it will be interesting to investigate whether the pre-established
ATOH1 targets are also get activated by this reprogramming strategy. Since it is
proposed that non-mammalian vertebrate supporting cells can de-differentiate into
progenitor-like cells before re-differentiate, it remains a challenge how to push the
mature supporting cells back to more sensory progenitor cell-like state.
75
Conclusion
In Chapter 2 and Chapter 3, I focused on our findings that (1) The utilization of 45% of
the de novo ATOH1 targets in the hair cells is dependent on the expression of Pou4f3 in
the hair cells, and (2) the hair cell specific distal regulatory elements, which are mostly
ATOH1 targets in hair cells, are decommissioned in the maturing supporting cells,
partially leading to the failure of their transdifferentiation. Our effort to understand the
development and the regeneration of inner ear hair cells generated more interesting
questions that need answers. Here I want to elaborate those questions, and at the same
time provide our hypothesis to these interesting questions.
1. Since the differentiation of the hair cells follows the basal to apical gradient, in a
non-synchronized manner, I think the 7,548 de novo hair cell specific distal
elements and the 3,264 ATOH1 targets among these regions are also created
(opened and activated) in a hierarchical manner. If we could do single cell
ATACseq analysis on the hair cells purified at different developmental stages, we
should be able to bioinformatically visualize the sequential activation of
thousands of the distal elements of interest. This analysis will also be useful to
understand the hierarchical expression of important transcription factors and their
targets during the differentiation of hair cells. So far the data in Chapter 2 only
provide the status of these enhancers at two developmental stages: E13.5 and
E17.5.
2. We demonstrated that around 45% of the 3,264 de novo ATOH1 targets are
POU4F3-dependent. One equally important question is what is the mechanism
76
that controls the utilization of the other ~55% of the de novo ATOH1 targets in
hair cells. The hypothesis is that there are other pioneer factors that work with
ATOH1 also in a feed-forward manner in hair cells, which controls the activation
of these distal regulatory elements. It will be interesting to know whether these
proposed transcription factors are also part of the proposed “conserved gene
regulatory network for mechanosensation”. I think Sox2 may be one of them.
3. In addition to the de novo ATOH1 targets, we found that there are 2,904 pre-
established ATOH1 targets, which are open and labeled by H3K4me1 in the
progenitor cells. We do not know during development how these pre-established
regions are programmed in the sensory progenitors. Nevertheless, I think these
pre-established, pre-existing distal elements are the pre-requisite for the de novo
distal elements. Although we have not make this point crystal clear in our
manuscript, we have shown a nice example: the pre-established Pou4f3
enhancers are the key to induce its expression, which leads to the utilization of
additional distal elements. Therefore the 11,133 pre-established hair cell specific
distal elements, which are not the focus of our current study, should contain
important information on the epigenetic landscape that are programmed to
become both hair cells and supporting cells in the inner ear.
4. In order to tell a short and coherent story about the ATOH1-POU4F3 feed
forward circuit, we made it look like that POU4F3 was created to be the best
friend of ATOH1. However, this is not true. First, more than 55% of the ATOH1
targets are not co-bound, or influenced by POU4F3. Second, about half of the
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POU4F3 dependent distal elements in hair cells are not bound by ATOH1. Third,
ATOH1 and POU4F3 are not sufficient for the opening of most of their targets. In
addition, the expression of Atoh1 is transient, but the expression of Pou4f3 in the
hair cells is permanent. Therefore, the mechanisms behind ATOH1/POU4F3-
mediated hair cell differentiation are very rich and interesting. Our unpublished
data show that around 5,000 distal elements are getting open in the maturing hair
cells, and that POU4F3 motifs are enriched in these regions. These regions have
nothing to do with ATOH1, since Atoh1 is not expressed at later stages.
Therefore, these observations suggest that pioneer factors like POU4F3 can
potentialize a reservoir of potential regulatory elements, the utilization of which is
dependent on the expression of other transcription factors, and the evolution of
which is subject to mutations followed by nature selection.
5. We claim, based on literature, that the hair cells in the mammalian inner ear do
not naturally regenerate. However, I do not think researchers have searched all
the mammals. People think mammalian skin do not regenerate large wound, until
they found that the African spiny mice have the capacity to fully regenerate large
piece of lost skin and the complete functions. It is unknown whether African spiny
mice can regenerate their hair cells. After hair cell loss in mice, it remains unclear
whether the supporting cells form “scar-like” tissue, which may prevent their
ability to proliferate and differentiate. Although this is a different hypothesis from
what we have shown in Chapter 3, it worth to examine the molecular identity of
the remaining supporting cells in the deafened adult mice.
78
Chapter 5: Experimental procedures
Animals
The experimental procedures were approved by the Animal Care and Use Committees
of University of Southern California. Cdkn1b-Gfp BAC transgenic (Lee et al. 2006; White
et al. 2006), Atoh1-Gfp knock-in (Rose et al. 2009), Pou4f3-Dtr knock-in (Tong et al.
2015), Atoh1-flox knock-in (Shroyer et al. 2007), and Pax2-Cre BAC transgenic
(Ohyama and Groves 2004) mouse lines were used in this study. Expression of
POU4F3 was disrupted in Pou4f3
Dtr/Dtr
animals due to the insertion of hDTR gene at the
start codon of Pou4f3 gene (Tong et al., 2015). POU homozygous mutant mice used in
this study were also Atoh1
Gfp/Gfp
. F-actin enriched stereocilia of the utricular hair cells
was stained by Phalloidin-iFluor (Abcam, ab176759; diluted 1/1,000 in PBS containing
0.05% Triton X-100), and the absence of stereocilia staining in Pou4f3
Dtr/Dtr
utricles
allowed fast identification of Pou4f3 homozygote mutant, from heterozygote mutant, and
wild type littermates (Hertzano et al., 2004; Xiang et al., 1998). Embryos with the Atoh1
gene conditionally knocked out were generated by crossing Atoh1
flox/+
; Pax2
Cre/+
animals
to Atoh1
flox/flox
animals.
Fluorescence-activated cell sorting
Cochlear tissue was incubated with trypsin (Invitrogen, 0.25%) at 37°C for 8 minutes. To
stop the reaction, FBS (Invitrogen) at a final concentration of 2% was added. The
tissues were dissociated into single-cell suspension by trituration with a p200 pipette for
3 min, and then filtered with 40μm VWR cell strainer. DAPI (1:10,000; Invitrogen) was
added to single-cell suspension to mark dead cells during FACS purification.
79
The skin tissue (head, limbs and tail removed) from E17.5 mouse embryos was
digested with 5mg/mL dispase II (Sigma, D4693) in PBS at 37 °C for 45 minutes. The
epidermal tissues were separated from dermis with forceps, and then incubated with
trypsin (Invitrogen, 0.25%) at 37 °C for 10 minutes. To stop the reaction, FBS
(Invitrogen) was added to a final concentration of 5%. The tissue was dissociated into a
single-cell suspension by trituration with P1000 pipette, and then passed through a
40μm VWR cell strainer. DAPI was added to mark dead cells during FACS purification.
Merkel cells were purified by their Atoh1-GFP expression. Touch dome epithelial
progenitors were FACS-purified by elimination of Merkel cells (GFP+), and staining for
progenitors with antibodies to Integrin-α6, Cd34, Sca-1, and CD200 as described
previously (Doucet et al., 2013).
Fluorescence-activated cell sorting was done on Aria II (BD Biosciences). Cells were
collected either into PBS with 5% FBS for µATACseq and C&R, or into RNA lysis buffer
(Zymo Research, R1060) for RNAseq. The purity of the target population was confirmed
to be >90% by re-sort, and/or cell counting.
μATACseq
μATACseq is based on ATACseq (Buenrostro et al., 2013), with some modifications,
including the elimination of the nuclear purification step. Hyperactive Tn5 was produced
as previously described (Picelli et al. 2014). The transposition activity of homemade Tn5
was assessed, and activity was defined as the amount of transposase that was able to
convert 100 ng λDNA to fragments of an average size of 200-400 bp in 15 minutes at
55˚C. FACS-purified cells (500 to 3000 cells per reaction) were centrifuged to remove
80
the supernatant. 20 μL of lysis buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF,
0.2% N-P40) was added, followed by pipetting 6-10 times to release the nuclei without
purification, and 30 μL reaction buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10%
DMF, 1000 unit transposome) was mixed on a Vortex for 5 seconds. The reaction was
incubated at 37 °C for 20 minutes, followed by addition of 350 μL ERC buffer (MiniElute,
Qiagen) to stop the reaction. After DNA purification (MiniElute, Qiagen), ATACseq
libraries were constructed as previously described (Buenrostro et al., 2013).
CUT&RUN (C&R)
Genome-wide histone modifications and transcription factor bindings were assayed by
the CUT&RUN technique according to the original protocol (Skene and Henikoff 2017).
Approximately 5,000 cells were used for histone modifications, and ~10,000 cells for
transcription factor binding studies. The following antibodies were used: rabbit-anti-
H3K4me1 (Active Motif, #61633), rabbit-anti-H3K27ac (Active motif, #39133), rabbit-
anti-H3K27me3 (Active Motif, #39155), rabbit-anti-H3K4me3 (Active Motif, #39159),
rabbit-anti-GFP (Torrey Pines Biolabs, TP401), mouse-anti-POU4F3 (Santa Cruz, sc-
81980), and secondary rabbit-anti-mouse antibody (Abcam, ab46540)
DNA extracted from CUT&RUN samples were made into sequencing libraries using
Accel-2S DNA library prep kit (Swift Biosciences) with MID and single index adapters.
DNA concentration and fragment distribution were measured on a Tapestation (Agilent),
using high-sensitive D5000 tape. Libraries were pooled to equal molarities and then
diluted to 4nM, for sequencing.
81
Analysis of μATACseq and CUT&RUN data
Libraries from μATACseq and C&R were sequenced using the NextSeq 500 platform
(Illumina) and a minimum of 20 million paired-reads/sample was collected. The Encode
analysis pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline) for ATACseq
and ChIPseq data, were modified and used to analyze our results. Briefly, the raw reads
were trimmed to 37bp and aligned to GRCm38/mm10 genome assembly by STAR
aligner (Dobin et al. 2013). PCR duplicates were removed based on genomic
coordinates for ATACseq, or by MIDs using UMI-tools for C&R, reads that aligned to
“blacklist regions” (Amemiya et al. 2019) were removed, and then peaks were called by
Model-based analysis of ChIP-Seq (MACS2) (Zhang et al. 2008) with p=0.01 cutoff and
disabled dynamic lambda option (--nolambda) for individual replicates. Peaks from
individual replicates were further filtered by IDR<0.01, and the overlapping peaks
between replicates were used. Bigwig files were generated from dup-removed bam files
with bedtools and bedgraphtobigwig, and normalization was based on total read
numbers. Heatmaps were generated with DeepTools (Ramírez et al. 2016) based on
the normalized bigwig signal files. Individual genomic loci were examined by IGV (Broad
Institute). HOMER (Heinz et al. 2010) was used to identify de novo motifs. Differential
analyses of ATACseq data was done with DESeq2 (Love et al. 2014). Gene ontology
analyses was done by EnrichR (Chen et al. 2013) and GREAT (McLean et al. 2010).
RNAseq
Total RNA was extracted from FACS-purified cells with Quick-RNA Microprep kit (Zymo
Research), quantified on a Tapestation (Agilent), and processed for library construction
82
with QIAseq FX Single Cell RNA Library Kit (Qiagen). At least 2 biological replicates
were collected for each sample/ condition, and over 20 million reads were sequenced
for each replicate. Reads were mapped to GRCm38/mm10 genome assembly by STAR
aligner (Dobin et al. 2013), and counted against transcript by htseq-count (Anders et al.
2015). Differentially expressed protein coding genes were identified using DESeq2
(Love et al., 2014). FPKM was calculated by Cufflinks (Trapnell et al. 2012).
In vitro overexpression of transcription factors in MEF
Mouse embryonic fibroblasts were obtained from E13.5 wild type embryos after
carefully removing the organs and the spinal cord. Tissue was minced and
enzymatically treated with 0.25% trypsin-EDTA for 30 minutes at 37°C. Trypsin
treatment was stopped by2% FBS (Invitrogen), and tissue was then dissociated by
trituration with P1000 pipette for 3 minutes. The dissociated cells were centrifuged
(800g for 10 minutes) and the pellet was suspended in MEF media (DMEM/F12 and
10% FBS) before plating in gelatin coated T75 tissue culture flasks. The MEFs were
expanded in culture and then cryopreserved in liquid nitrogen using freezing media
(MEF media plus 10% DMSO). MEFs were cultured to reach 70-80%% confluency
before lentivirus infection. All cell cultures were tested negative for mycoplasma
contamination.
Virus was produced by transfection of HEK293 with 3 constructs, pPAX2, pVSVG, plus
pHR plasmids containing the coding regions for alternately, ATOH1-t2a-GFP, POU4F3-
p2a-tdTomato or GFP control (sequences upon request) using FUGENE HD
(Promega). Three days after transfection, cells and media were harvested, filtered
83
(0.45µm filter, Sigma). The supernatants containing the lentiviruses were used without
further concentration steps.
Organotypic Utricular Culture
Utricles were dissected from postnatal day 2 pups (Atoh1
flox/flox
;Pax2
Cre/+
mice) in pre-
chilled PBS. The utricular sensory epithelia in Atoh1 conditionally-mutant mice were
identified morphologically (Bermingham et al. 1999), and retained only sparse
innervation at this stage (Fritzsch and Beisel 2004), compared to wild type. The utricles
from Atoh1 mutant animals were mounted on SPI black membranes (SPI Supplies)
floating on culture medium (DMEF/F12, supplemented with 100U penicillin, 1:100 N2
(Invitrogen), 1:50 B27 (Invitrogen), 5 ng/ mL EGF (Sigma) and 2.5 ng/ mL FGF (NIH)),
and cultured at 37 °C, 5% CO2 and 5% O2, as described previously (Doetzlhofer et al.
2009).
AAV used for utricular infection was produced as previously described (Suzuki et al.
2017). Three plasmid constructs were transfected into HEK293 cells using FUGENE HD
(Promega). These plasmids were dF6 adenoviral vector, pAAV2/ Anc80L65 encoding
AAV2 rep and Anc80L65 cap genes, and a plasmid containing the ITR-sequences
flanking the coding regions for ATOH1-t2a-GFP or GFP control. Three days after
transfection, cells and media were harvested and filtered. The AAV virus in the
supernatant was concentrated using AAVanced Concentration reagent (SBI, AAV100A).
Concentrated AAV-ATOH1-t2a-GFP, or AAV-GFP control virus was added to the
culture media. Tissues were fixed 72 hours post-infection for immunohistochemistry.
Immunohistochemistry
84
Epidermis preparation: Skin was prepared from E17.5 mouse embryo trunk regions
(head, limbs and tail removed) and treated with 5mg/mL dispase II (Sigma, D4693) in
PBS at room temperature for 20 minutes. The epidermis was carefully separated from
dermis using forceps, then cut into small pieces. Tissue was fixed with 4%
paraformaldehyde in PBS at room temperature for 15 minutes. Subsequently, the
tissues were blocked and permeabilized overnight at 4 °C with 10% donkey serum
(Millipore, S30-100ML) in PBS, supplemented with 0.5% Triton X-100 (Sigma, T8787).
Primary and secondary antibody incubations were performed sequentially overnight at 4
°C in 1% donkey serum in PBS, supplemented with 0.05% Triton X-100. Washing with
PBS at room temperature for 15 min was performed twice after each antibody
incubation. The epidermal tissue was mounted on slides with the basal layer facing up.
Cochleae preparation: from E13.5-E15.5 embryos were collected in pre-chilled PBS
after dissection. The organs were incubated in PBS containing 1mg/mL dispase II
(Sigma, D4693) and 1mg/mL collagenase (Worthington) at 37 °C for 8 minutes, and
then cochlear ducts were freed from surrounding mesenchyme using forceps, as
previously described (Doetzlhofer et al. 2009). Cochleae from E17.5 or older
embryo/pups were dissected from inner ears in pre-chilled PBS. The spiral ganglia,
Reissner’s membrane, and the lateral wall were carefully removed to obtain cochlear
surface preps. Cochlear ducts or surface preps were fixed and stained with the
aforementioned protocol..
Primary antibodies were diluted at 1:500, and secondary antibodies were diluted at
1:5,000. Primary antibodies used in this study were mouse anti-p27
kip1
(Thermo Fishers,
85
AHZ0452), rabbit anti-MYO7A (Proteus BioSciences, 25-6790), chicken anti-GFP
(GeneTex, GTX13970), mouse anti-POU4F3 (Santa Cruz, sc-81980), goat anti-SOX2
(Santa Cruz, sc-17320, discontinued), mouse anti-KRT20 (Dako, M701929-2), rabbit
anti-Caspase3 (R&D, AF835), mouse anti-LHX3 (DSHB, 67.4E12), rabbit anti-SIX1
(Cell Signaling, 12891s). Secondary antibodies used in this study were from Thermo
Fishers or Jackson ImmunoResearch.
86
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Appendix 1: μATACseq validation (see method)

103
Appendix 1 (continued): μATACseq validation

Using μATACseq to profile chromatin accessibility with as few as 100 cells. (A)
Genome browser representation of the comparison of data collected from the standard
ATACseq and μATACseq with different cell numbers. Data collected from 100 cells with
μATACseq protocol look almost identical to those from 20,000 cells with the standard
ATACseq protocol. (B) Pearson correlation among the data collected from the standard
ATACseq and μATACseq with different cell numbers. (C) Histogram representation of
the read distribution comparison of data collected from the standard ATACseq and
μATACseq with different cell numbers shows similar proportions of reads falling in
promoters, enhancers and non-peak regions. (D) Scatter plot shows how many percent
of the standard ATACseq peaks are detected from μATACseq with different cell
numbers. In the 100-cell group, more than 70% of the top 50,000 peaks from the
20,000-cell group were detected. (E) Comparison of the number of uniquely mapped
reads, percentage of mitochondria reads and the percentage of duplicated reads of data
from the standard ATACseq and μATACseq with different cell numbers. (F) Comparison
of the read length distribution among the data from the standard ATACseq and
μATACseq with different cell numbers. On average, the reads became shorter as the
cell number decrease, because less cells means higher enzyme-to-cell ratios, which
leads to more complete digestion and shorter reads. However, no over-digestion was
observed. G) Comparison of the average footprint depth at the predicted CTCF binding
sites among the standard ATACseq and μATACseq with different cell numbers. In the
100-cell group, we observed a slightly shallower average footprint at CTCF binding
sites, compared to the other groups, consistent with the higher enzyme-to-cell ratio.
Nevertheless, the footprints from all four groups were clear and informative, indicating
that our protocol is robust with different enzyme-to-cell ratios without causing over-
digestion.

Abstract (if available)

Abstract Neurosensory cells are epithelial cells with neuronal characteristics including being excitable by both internal and external stimuli. In vertebrates, neurosensory cells such as hair cells, gustatory cells, and Merkel cells are specialized for the senses of hearing, balance, taste and light touch. Aside from these, there are many types of neuroendocrine cells in the internal organs. For example, pulmonary neuroendocrine cells (PNECs) in the airway epithelium and enteroendocrine cells in the gut collaborate with immune cells and neural circuits, in order to respond to signals in the inhaled air or ingested contents. Despite their different embryonic origins and their division of labor in perceiving stimulants, these neurosensory cells share a striking array of similarities at the cellular and molecular level, including the expression of ion channels, neurotransmitters, synaptic proteins, cilium components, etc. ❧ Hearing and balance are mediated in vertebrates by inner ear mechanosensory hair cells. Hair cell development, maturation, and survival require the expression of ATOH1, a bHLH transcription factor that is considered a master regulator of hair cell differentiation. However, we now show that at the time of hair cell differentiation, a large percentage of the ATOH1 targetome lies in “closed” chromatin, to which ATOH1 is unable to bind. We show that one of the first direct targets of ATOH1 expressed in hair cells is POU4F3, a class IV POU-domain transcription factor that is also necessary for hair cell differentiation. ATOH1 can bind to several open enhancers at the Pou4f3 locus, and activates its early expression in hair cell precursors. Unlike ATOH1, we show that POU4F3 can bind “closed”, nucleosomal DNA, and has pioneer factor activity needed to remodel the closed ATOH1 targetome, allow access to ATOH1, and thereby promotes hair cell differentiation in a feed-forward manner. We demonstrate that this feed-forward mechanism is also necessary for the differentiation of Merkel cells, a mechanosensory cell population in the skin responsible for mediating light touch. Although the transcriptomes of hair cells and Merkel cells are very different, they share many POU4F3 and ATOH1 chromatin targets, suggesting that different vertebrate mechanoreceptors may use elements of the same ancient epistatic gene regulatory mechanism, supplemented by distinct enhancer networks, to produce specific mechanoreceptive cell types. ❧ One of the striking differences between the hair cells and the other secondary receptor cell types in mammals is regenerative capability. No adult stem cells are present in the mammalian inner ear, and death of mechanosensory hair cells fails to elicit regeneration from the surrounding supporting cells. In contrast, supporting cells in non-mammalian vertebrates are able to divide and transdifferentiate into hair cells to restore function following hair cell loss. A latent potential for supporting cell direct transdifferentiation can be elicited in the neonatal organ of Corti of mice by blocking Notch-mediated lateral inhibition, suggesting that the gene regulatory network governing transdifferentiation remains intact in perinatal supporting cells. By profiling chromatin accessibility and histone modifications in mouse hair cells and supporting cells, we show that hair cell gene enhancers primed by H3K4me1 are silenced in supporting cells by H3K27 trimethylation and active deactylation. These marks are rapidly reversed during neonatal supporting cell transdifferentiation in response to Notch inhibition. However, this latent transdifferentiation potential is lost by the end of first postnatal week. We report that loss of transdifferentiation potential is accompanied by a loss of H3K4me1-priming at hair cell gene enhancers in supporting cells. We further show that blocking H3K4me1 removal leads to a temporal extension of transdifferentiation potential. We hypothesize that this enhancer decommissioning contributes to the failure of supporting cell regeneration in the mature organ of Corti.

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University of Southern California Dissertations and Theses

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Creator Yu, Haoze (author)

Core Title Transcriptional and epigenetic mechanisms underlying sensory hair cell differentiation and regeneration

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Publication Date 09/17/2020

Defense Date 08/18/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Atoh1,feed-forward,hair cells,Merkel cells,OAI-PMH Harvest,pioneer factor,Pou4f3

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McMahon, Andrew (committee chair), Crump, Gage (committee member), Segil, Neil (committee member)

Creator Email haozeyu@usc.edu,vincentyu8901@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-373479

Unique identifier UC11666604

Identifier etd-YuHaoze-8960.pdf (filename),usctheses-c89-373479 (legacy record id)

Legacy Identifier etd-YuHaoze-8960.pdf

Dmrecord 373479

Document Type Dissertation

Rights Yu, Haoze

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

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