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DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration

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DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration

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Content DNA methylation in the mouse cochlea promotes maturation of supporting cells and
contributes to the failure of hair cell regeneration
by
John Duc Nguyen
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(DEVELOPMENT, STEM CELLS, AND REGENERATIVE MEDICINE)
May 2023
Copyright 2023 John Duc Nguyen
ii
Dedication
This thesis is first dedicated to my PhD mentor Neil Segil, who has warmly
welcomed me into his lab and nurtured both my scientific curiosity and development as
a citizen. I approached Neil with only an interest in learning bioinformatics and Next
Generation Sequencing (NGS) technologies, and he was immediately welcoming to me
joining the lab. At that point in my life, I did not even know where the ear was located on
the human body, let alone the inner ear! Neil had a philosophy that whether you were
interested in the biology or the technology, it was more important that you were even
interested at all, and it was that self-driven motivation to learn that was the
quintessential trait that Neil was looking for. Through time and dedication to a project,
Neil believed that our feelings would grow for the field he dedicated his life to, inner ear
biology and regeneration. I am glad to have had you as my mentor and I miss you, Neil.

iii
Acknowledgements
What a crazy, wonderful, amazingly tumultuous 6 years. I never thought a PhD
training would involve so much philosophical and psychological expeditions within
myself. After it all, I feel like I have lived an entire life. Through its many up’s and
down’s, long nights, and innately stressful situation, I think I have learned just as much
about who I am as a person, as I have about inner ear biology. I’ve grown so much as a
scientist and as an individual through the relationships I’ve developed during my time
here at USC Keck School of Medicine. I would like to take the following section to give
thanks to everyone that contributed to this experience along the way. For the sake of
brevity, I only thank the immediate members of the Segil Lab and my mentorship
committee. If I missed you, you already know who you are, gold chain gang.
I thank Neil for giving me the room to grow and explore my own scientific ideas. I
thank Neil for providing a great work environment and leading a team that felt more like
a family. I thank Neil for setting out time to talk to me about science, life, and everything
in-between. I will the moments where we quoted Robert Frost poems to each other.
There is a verse in “Two Tramps in Mud Time” by Robert Frost that I think Neil wished
to impart on all his mentees:
But yield who will to their separation,
My object in living is to unite
My avocation and my vocation
As my two eyes make one in sight.
Only where love and need are one,
iv
And the work is play for mortal stakes,
Is the deed ever really done
For Heaven and the future's sakes.
As Neil has found love in his science, I, too, am finding my own passion thanks to his
mentorship and guidance.
I would like to also dedicate this thesis to the Segil Lab members. Juan Llamas,
my partner in crime, has not only helped mentor me as a scientist, but also pushes me
out of my comfort zones, whether we are scaling Mount Whitney or training at the gym
together. Welly Makmura, a friend and a mentor as well. Both Juan and Welly were
great confidants and provided great counsel about life and its many philosophies. Thank
you, Litao Tao and Vincent Haoze Yu for all the great times we had in the outdoors. I
never thought that I find myself fighting turbulent ocean waves on a small kayak to fish
for lobsters with lab members! Thank you, Talon Trecek for always bringing “data” to
mixers, and keeping my college years alive. We went to a good bit of music events
together. Thank you, Frankie James for sharing with us your homebrewed beers and
entertaining us with your unicycle antics. Thank you Xizi Wang for introducing me to so
many great restaurants. Thank you, Jennifer Rubio for sharing all your cat stories.
Thank you Ksenia Gnedeva for showing me that postdocs can still have a great time!
Thank you, Robert for your immaculate attention to detail on PowerPoint slides. Thank
you, Tuo Shi for making my last ARO Midwinter Meeting memorable; Disneyland was
fun! Thank you, Leah Kim for instigating all the social gathering events at Korean
restaurants. Thank you, Nathan Segil for nerding out with me over LEGOs and art.
v
Thank you, Louise Menendez and Van Haslett for contributing to all the fun. Lastly, I
almost forgot to thank everyone for the superb science. Segil Lab! Segil Lab! Segil Lab!
Thank you to the mentors who stepped in to help me finish my PhD after Neil’s
passing. Andrew McMahon helped maintain the infrastructure, provided guidance and
professional mentorship. Andrew Groves helped with paper writing and submission, as
well as professional mentorship.
I would like to also thank my family for supporting me during my 6 years of PhD
training. I understand that as a first-generation Vietnamese America, there is a great
weight of responsibility that falls upon my shoulders to provide for my family and change
our means for the better. Having decided to not attend medical school, I know it must
have been worrisome to see your son pursue a degree that carries with itself a lot more
financial uncertainty. The job description that I would tell you was that I “worked with
mice,” which might cause some alarm, considering that other professions such as
doctor, lawyer, and engineer seemed to be intuitively more practical for society.
However, as I near the end of my PhD training, I can see your confidence has grown in
the choice that I have made, as I am getting job offers and attending conferences to put
our family name out there. I have debated long and hard about which path to take next,
whether I should stay in academia or move on to industry. I still understand my
responsibilities to you, my family, to secure a financial future. Therefore, I have decided
to take the industry route. Maybe someday, the future generation may be well off
enough to entertain a career in academia. Thank you to Peter Minh Nguyen, my dad, for
providing me with this opportunity and the financial stability to focus on my schooling. I
vi
understand you have sacrificed a lot to get us here. Thank you to Trinh Thi Tu Pham,
my mom, for providing me with unconditional motherly love. Stereotypically Asian mom,
you cooked and froze down so much food to fuel me during my schooling, even well
after I have left the nest. I love you both, mom and dad. I would also like to give a
shoutout to Thuy Le, Jason The Minh Nguyen, Mason Khang Minh Nguyen. Y’all were
awesome and motivated me to set some precedence and break down walls for you to
follow in my path.
Lastly, I would like to thank my significant other, Anne Dieu An Nguyen, for being
my rock during the last year of my PhD. Thank you so much for consoling me and
providing the support for me to push forward. Your paper revisions were a life-saver; I
am always awed by your literary acumen. You also kept me sane and helped me
maintain perspective when the road got rocky. I couldn’t have done it without you.

vii
Table of Contents
Dedication ......................................................................................................................... ii
Acknowledgements .......................................................................................................... iii
List of Tables .................................................................................................................... x
List of Figures .................................................................................................................. xi
Abstract .......................................................................................................................... xiii
Chapter 1. Epigenetic regulation of cochlear sensory epithelia development ................. 1
Introduction ................................................................................................................... 1
Background and Significance ..................................................................................... 12
Chapter 2. NuTRAP reporter mice allow isolation of nuclei from the
postnatal cochlea for epigenetic profiling ....................................................................... 17
Introduction ................................................................................................................. 17
Mechanical and detergent-based lysis of cochlea epithelia releases
high quality nuclei for downstream NGS applications ................................................. 19
Using the NuTRAP transgenic mouse to label nuclei of target cell populations ......... 26
Chapter 3. Epigenetic maturation of organ of Corti supporting cells and the
hair cell gene regulatory network ................................................................................... 32
Introduction ................................................................................................................. 32
Cochlear progenitor cells, hair cells and supporting cells have distinct DNA
methylation signatures. ............................................................................................... 33
DNA methylation signatures highlight gene regulatory network switching
within the prosensory epithelium as it matures. .......................................................... 40
DNA methylation encroachment and heterochromatinization of
pre-established CpG island promoters occurs in postnatally maturing
supporting cells. .......................................................................................................... 42
Chapter 4. DNA methylation in the mouse cochlea contributes to the
failure of hair cell regeneration ....................................................................................... 51
Introduction ................................................................................................................. 51
DAPT-mediated transdifferentiation induces de novo DNA demethylation of
hair cell-specific DMRs ............................................................................................... 51
viii
Chapter 5. The epigenetic effects of long-term deafening on
adult mouse cochlear supporting cells ........................................................................... 59
Introduction ................................................................................................................. 59
Simultaneous profiling of RNA and ATAC at the single cell level in
cochlea of wildtype and long-deafened mice .............................................................. 60
Conclusion ..................................................................................................................... 69
Materials and Methods ................................................................................................... 75
Animals ....................................................................................................................... 75
Epithelial preparation for E13.5 cochleas ................................................................... 76
Sensory epithelium dissection for P1, P6, P8, P21, and P70 cochleas ...................... 76
Cochlear explant culture and drug treatment .............................................................. 77
Single cell dissociation for E13.5, P1, P8, and P21 cochleas ..................................... 77
Nuclei isolation for P6, P8, P21, and P70 cochleas .................................................... 78
FACS purification ........................................................................................................ 79
Cryosectioning, Immunostaining, and microscopy ..................................................... 80
Whole genome bisulfite sequencing (WGBS) library preparation ............................... 80
CUT&RUN .................................................................................................................. 81
CUT&TAG ................................................................................................................... 82
P70 wildtype and long-term deafened mouse model for scMultiome ......................... 83
scMultiomic (simultaneous scRNA-seq and scATAC-seq profiling)
sample processing ...................................................................................................... 83
scMultiomic data processing ....................................................................................... 84
Generation of “pseudobulk ATAC” signal from scMultiome ........................................ 85
NGS Alignment and Data Analysis ............................................................................. 86
References ..................................................................................................................... 89

x
List of Tables
Table 3.1. Homogenization Buffer ................................................................................. 23
Table 3.2. Artificial Cerebral Spinal Fluid (ACSF) .......................................................... 23
Table 3.3 Nuclear extraction buffer (NEB) ..................................................................... 24

xi
List of Figures
Figure 1.1. Mechanosensory hair cells within the organ of Corti fail to functionally
regenerate. ....................................................................................................................... 2
Figure 1.2. Cochlear supporting cells share a common progenitor, but
fail to regenerate into hair cells postnatally. ..................................................................... 3
Figure 1.3. Notch-mediated lateral inhibition to direct hair cell and
supporting cell fate decisions ........................................................................................... 5
Figure 1.4. Epigenetic chromatin states can be divided into poised, active, and
heterochromatin. .............................................................................................................. 8
Figure 1.5 DNMT inhibitor RG108 increases percentage of supporting cell
transdifferentiation in response to DAPT treatment. ...................................................... 15
Figure 1.6. DNMT inhibitor RG108 increases percentage of supporting cell
transdifferentiation in response to DAPT treatment. ...................................................... 16
Figure 2.1 Morphological changes of organ of Corti during postnatal maturation. ........ 18
Figure 2.2. Nuclei separation from cell debris. ............................................................... 24
Figure 2.3.Enzyme-based dissociation versus dounce homogenization of
cochlear tissue. .............................................................................................................. 25
Figure 2.4. NuTRAP transgenic mouse labeling system of nuclei ................................. 29
Figure 2.5. Atoh1-creER NuTRAP mouse labels hair cells of the
cochlea and utricle ......................................................................................................... 30
Figure 2.6. NuTRAP nuclei after dounce homogenization and iodixanol gradient
separation. ..................................................................................................................... 30
Figure 2.7. FACS purification of NuTRAP labeled nuclei ............................................... 31

Figure 3.1. Quality control analysis of WGBS datasets depicts typical
characteristics of mCpG distribution .............................................................................. 34
Figure 3.2. Distinct DNA methylation patterns are enriched at select hair cell and
supporting cell enhancers .............................................................................................. 37
Figure 3.3. Pre-established CGI promoters exhibit DNA methylation encroachment
and a silenced chromatin state in maturing supporting cells ......................................... 46
xii
Figure 3.4. H3K27me3 signal is comparable across samples and is
inversely correlated to DNA methylation. ....................................................................... 49
Figure 3.5. Characterization of H3K27me3, H3K4me1, and CUTAC signal at
genomic features. .......................................................................................................... 50

Figure 4.1. DAPT-mediated transdifferentiation of SCs induces de novo
hypomethylation of HC-specific enhancers. ................................................................... 55
Figure 4.2. Effect of DAPT-mediated transdifferentiation on
POU4F3 expression and DNA methylation signal at DMRs. ......................................... 58
Figure 5.1. Single cell multiomic profiling of mouse cochlear cell types
during developmental, adult homeostatic, and deafened states………………………….64
Figure 5.2. Integrating multimodal single cell datasets between RNA and ATAC,
and between P70 wildtype and long-deafened conditions. ............................................ 65
Figure 5.3. Single cell ATAC-seq profiling of key hair cell and
supporting cell genes. .................................................................................................... 66
Figure 5.4. ATAC signal shifts to a more accessibility state in long-term deafened
supporting cells compared to wildtype at hair cell-specific enhancers. .......................... 67
Figure 5.5. Linkage between accessibility and gene expression of Myo7a gene
show increased expression in deafened state compared to wildtype. ........................... 68

xiii
Abstract
Mammalian hair cells do not functionally regenerate in adulthood. However, in
mice, hair cells are regenerated at embryonic and neonatal stages in mice by direct
transdifferentiation of neighboring supporting cells into new hair cells. Previous work
showed loss of transdifferentiation potential in supporting cells is in part due to
H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during
the first postnatal week. However, inhibiting this decommissioning only partially
preserves transdifferentiation potential. Furthermore, the loss of H3K4me1 can be
considered as the loss of the “gas” for hair cell gene programs, but the question of the
“brakes” is unanswered and the focus of this thesis. Repressive epigenetic
modifications may serve as barriers to regulate the loss of cellular plasticity. For
instance, H3K27me3, a methylation of the H3 histone tail by PRC2, is a commonly
studied repressive epigenetic modification. The repressive H3K27me3 increases over
the hair cell gene regulatory network regions in postnatally maturing supporting cells as
enhancer decommissioning occurs through the loss of H3K4me1. This thesis is an
extension of this work, looking at DNA methylation, which is associated with a more
permanent form of epigenetic silencing through the physical compaction of chromatin
into a transcriptionally inactive state.
I hypothesized that once cochlear hair cells and supporting cells resolve their cell
fate decisions from their common progenitor origins, temporary repressive H3K27me3
modification will be replaced by DNA methylation or H3K9me3, solidifying the chromatin
structure and locking in either a hair cell or a supporting cell lineage. My study shows
xiv
supporting cells progressively accumulate DNA methylation at developmentally
regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of
Atoh1, a key transcription factor for hair cell fate, as well as many other developmentally
regulated genes involved in both hair cell differentiation and progenitor cell specification
and competency, which may explain why supporting cells are resistant to
reprogramming efforts and unable to upregulate the hair cell gene regulatory network.
Further, DNA hypermethylation of hair cell genes in supporting cells replaces
H3K27me3-mediated repression and is accompanied by a progressive loss of
chromatin accessibility, suggestive of facultative heterochromatin formation.
Interestingly, a subset of hair cell-specific differentially methylated regions (DMRs) was
only demethylated in hair cells and not in supporting cells. We hypothesized that these
regions act as a barrier to spontaneous transdifferentiation of supporting cells into hair
cells. If so, these regions must become demethylated for supporting cells to adopt a hair
cell phenotype. To address this prediction, we used DAPT-mediated Notch pathway
inhibition in P1 and P6 cochlear explant cultures to show that if we induce P1 supporting
cells to transdifferentiate into hair cells, transdifferentiating cells will demethylate hair
cell-specific DMRs through a TET-dependent demethylation process. Finally, we
observe changes in chromatin accessibility of supporting cell subtypes at the single cell
level with increasing age. Chromatin accessibility is lost at genes associated with
regulatory programs promoting sensory epithelium development loses chromatin
accessibility and gained in favor of gene programs promoting physiological maturation
and cochlear function. Surprisingly, chromatin accessibility is partially recovered in
xv
chronically deafened mouse model, an observation that holds promise for future
translational efforts in hearing restoration.

1
Chapter 1. Epigenetic regulation of cochlear sensory epithelia
development

Introduction
The most common cause of hearing loss is the damage and death of
mechanosensory cochlear hair cells, which do not functionally regenerate in adult
mammals (Figure 1.1).(Frolenkov et al., 2004; Furness, 2015; Ryan, 2000; Wagner &
Shin, 2019; Wong & Ryan, 2015). However, non-mammalian vertebrates such as
zebrafish and birds can regenerate hair cells throughout life, which occurs through
transdifferentiation of the surrounding supporting cells to mechanosensory hair cells,
with or without cell division (Figure 1.2A) (Balak et al., 1990; Corwin & Cotanche, 1988;
Cotanche, 1987; Cruz et al., 1987; Harris et al., 2003; Ryals & Rubel, 1988). In contrast,
mammals have only a transient potential for this transdifferentiation early within the first
perinatal week before the onset of hearing by postnatal day 10 (Korrapati et al., 2013;
White et al., 2006; Yamamoto et al., 2006; Zhao et al., 2011). Beyond this stage, which
in mice is just a few days after birth, the mammalian cochlea cannot functionally
regenerate hair cells once the hair cells have been killed (Kelly et al., 2012; Z. Liu et al.,
2012; Maass et al., 2015). Transdifferentiation of supporting cells into hair cells is an
attractive strategy for hair cell regeneration because both supporting cells and hair cells
arise from a common Sox2+ progenitor population within the prosensory epithelium
(Figure 1.2B) (P. Chen & Segil, 1999; Z. Liu et al., 2012; Maass et al., 2015). In fact, it
has been demonstrated that Sox2 activity

2

Figure 1.1. Mechanosensory hair cells within the organ of Corti fail to functionally
regenerate.
(A) Mechanosensory hair cells within the organ of Corti are arranged as one row of
inner hair cell and three rows of outer hair cells. These hair cells are interdigitated by
supporting cells below. Adapted from (Frolenkov et al., 2004) (B) Hair cells begin to die
off after exposure to ototoxic levels of Gentamicin. V-shaped hair bundles disappear as
hair cells die. Adapted from (Ryan, 2000).

3

Figure 1.2. Cochlear supporting cells share a common progenitor, but fail to regenerate
into hair cells postnatally.
(A) Non-mammalian vertebrates such as the chick demonstrates the ability to
regenerate hair cells from supporting cells through both mitotic and direct
transdifferentiation mechanisms throughout life. Mammalian vertebrates such as the
mouse fails to regenerate any functional hair cells in adulthood. (B) Both supporting
cells and hair cells are derived from a common Sox2+ progenitor population, and
mediated by Notch lateral inhibition to achieve a salt-and-pepper patterning. Adapted
from (Kelley & Stone, 2017).

4
is required to specify the prosensory domain at E12 prior to any hair cell or supporting
cell formation, and without Sox2, no hair cells or supporting cells are able to form due to
loss of competency (Gnedeva & Hudspeth, 2015). Following Sox2 competency
establishment, hair cell differentiation begins at E14.5 in mice with the onset of ATOH1
expression, a bHLH transcription factor that is both necessary and sufficient to promote
hair cell fate and survival (Bermingham et al., 1999; Cai et al., 2013; P. Chen et al.,
2002; Driver et al., 2013). Initially, ATOH1 is broadly expressed in the p27
kip1+
postmitotic prosensory progenitors of the cochlear epithelium (Cai et al., 2013; Li et al.,
2022; Woods et al., 2004). Subsequent upregulation of the Notch ligands DLL1 and
JAG2 by differentiating hair cells triggers Notch-mediated lateral inhibition of Atoh1
expression via Hes and Hey factors in adjacent cells and directs them towards a
supporting cell fate (Abdolazimi et al., 2016; Kiernan et al., 2005; Lanford et al., 1999).
The Sox2+ prosensory progenitor population at embryonic day 12 then becomes
the Sox2+ supporting cell population as a naming convention to distinguish from the
Atoh1+ hair cells once they have been specified (Dabdoub et al., 2008). However, there
is debate about whether the Sox2+ supporting cell population should still be considered
progenitor cells or even stem cells of the cochlea. Furthermore, Sox2+ post-mitotic
supporting cells have been shown to demonstrate some plasticity to reenter cell cycle
and proliferate and transdifferentiate into hair cells when they are dissociated from the
sensory epithelia and cultured (White et al., 2006). These studies suggest that at least
early postnatal supporting cells retain intrinsic ability to adopt a hair cell fate, and
require Notch lateral inhibition to stabilize their supporting cell fate commitment, making
5

Figure 1.3. Notch-mediated lateral inhibition to direct hair cell and supporting cell fate
decisions
(A) The future hair cell expresses Atoh1, the master transcription factor of hair cell fate,
as well as Notch ligands Jag2 and Dll1, which interact with the Notch1 receptor in
adjacent cells. This triggers cleavage and nuclear localization of the Notch intracellular
domain (NICD), which turns on Hes and Hey factors to repress Atoh1 in the adjacent
cells, pushing them towards a supporting cell fate. (B) Blocking Notch lateral inhibition
signaling will derepress Atoh1 in the adjacent supporting cells, and cause them to
transdifferentiate into hair cells. Adapted from Litao Tao.

6
them the ideal cell type to target reprogramming into hair cells (Korrapati et al., 2013;
Maass et al., 2015; Yamamoto et al., 2006).
During the first postnatal week, mouse cochlear supporting cells retain some
plasticity to transdifferentiate into hair cells, suggesting they may be maintained in a
progenitor-like state (Bramhall et al., 2014; Chai et al., 2011, 2012; Shi et al., 2012;
Sinkkonen et al., 2011). The transdifferentiation process can be initiated by killing hair
cells (Cox et al., 2014), or by disrupting Notch-mediated lateral inhibition, which
derepresses ATOH1 (Figure 1.3) (Doetzlhofer et al., 2009; Maass et al., 2015; Zhao et
al., 2011). However, this plasticity to transdifferentiate is lost by postnatal day 6 (Kelly et
al., 2012; Z. Liu et al., 2012; Maass et al., 2015). During this period, the maturing mouse
cochlea undergoes dramatic remodeling of the extracellular matrix, cytoskeleton, and
gap junctions before hearing onset at P14, reflecting a transition from a program that
promotes sensory development to one that promotes physiological maturation and
function (Lim & Anniko, 1985; Tritsch & Bergles, 2010; Walters et al., 2017; H. C. Wang
et al., 2015).
This change from a state that maintains regenerative ability to a state of
functional maturation is driven by both transcriptional and epigenetic changes (Maass et
al., 2016; Stojanova et al., 2015). The Segil Lab previously showed that one mechanism
that partly explains the loss of transdifferentiation potential is the decommissioning of
hair cell enhancers in maturing supporting cells marked by H3K4me1 removal (Tao et
al., 2021). This enhancer decommissioning resolves bivalent or poised hair cell
enhancers in supporting cells, where the enhancers were initially marked by both the
7
permissive H3K4me1 and H3K27me3 repressive histone modifications. In the default
hair cell lineage, these hair cell enhancers would resolve by swapping H3K27me3 in
favor of H3K27ac to promote the activation of these enhancers and their target
promoters and genes, while retaining H3K4me1. In the secondary supporting cell
lineage, Notch lateral inhibition prevents the master transcription factor of hair cell fate
Atoh1 from being expressed, whatsoever, so the hair cell-specific enhancers are
maintained in a poised but repressed epigenetic state. Then by postnatal day 6, these
hair cell enhancers become actively decommissioned, potentially through LSD1
H3K4me1-specific demethylase activity (Tao et al., 2021). It has been shown that
H3K4me1 plays a role in poising the enhancer for activation during developmental gene
regulation and differentiation in a cell-type and stage-specific expression pattern
(Creyghton et al., 2010; Rada-Iglesias et al., 2011).
However, other repressive epigenetic mechanisms such as heterochromatin
formation are known to further silence genes of alternative cell identities, and reinforce
cell fate commitment during development and maturation (Figure 1.4) (Becker et al.,
2016; Matoba et al., 2014; Soufi et al., 2012; Trojer & Reinberg, 2007; Wallrath & Elgin,
1995).These include H3K9me3, and DNA methylation which involves the methylation of
cytosines in CpG dinucleotide context, which together promotes the physical
compaction of chromatin into what is known as heterochromatin (Becker et al., 2016;
Rountree & Selker, 2010; Trojer & Reinberg, 2007). Heterochromatin is a tertiary higher
order of chromatin organization, where the chromatin embedded within are completely
condensed and refractory to transcriptional machinery. It has even been suggested that
8

Figure 1.4. Epigenetic chromatin states can be divided into poised, active, and
heterochromatin.
Heterochromatin is characterized by the methylation, or addition of a methyl group, onto
cytosines within a CpG dinucleotide sequence in DNA, along with tri-methylation of the
H3 histone tail, at the lysine 27 position, H3K27me3. Heterochromatin are silenced
regions of chromatin and tend to be inaccessible by ATAC-seq. Poised enhancers are
characterized by accessibility by ATAC-seq, H3K4me1, a priming mark, and
H3K27me3, a repressive mark. Active enhancers swap H3K27me3 repression for
H3K27ac activation. Adapted from (Ordoñez et al., 2019).

9
heterochromatin shuts down chromatin by phase separation from regions of active gene
transcription in the nucleus (Larson & Narlikar, 2018; Tachibana et al., 2007; H. Zhang
et al., n.d.). Together H3K9me3, DNA methylation, and their respective readers and
writers G9a, Suv39H1, DNMT3a, DNMT3b, and HP1 positively reinforces the other to
solidify the chromatin compaction state (Chang et al., 2009; Chiba et al., 2015; Du et al.,
2015; Fuks et al., 2003; Liao et al., 2015; Vermeulen et al., 2010). In addition to this
tightly regulated positive feed-forward mechanism, H3K9me3 also tethers the
heterochromatin regions to the nuclear periphery (Harr et al., 2015; Lienert et al., 2011;
Luo et al., 2009; Peric-Hupkes et al., 2010; Poleshko et al., 2017), where transcriptional
output tends to be lower compared to the nuclear core territory (Briand & Collas, 2020;
S. Chen et al., 2018).
Within the context of supporting cell maturation and loss of plasticity to
transdifferentiate into hair cells, we hypothesized that hair cell genes are further
silenced in supporting cells by heterochromatin formation during their maturation. For
instance, key hair cell gene regulatory network such as enhancers downstream of
transcription factors like Atoh1, Pou4f3, Gfi1, or Gata3 may be entombed within
heterochromatin regions and completely inaccessible to these transcription factors,
regardless of their forced expression in reprogramming efforts (Iyer et al., 2022;
Menendez et al., n.d.; Soufi et al., 2012). To study the possibility of heterochromatin
formation, we decided to first look at DNA methylation at CpG dinucleotides, which is
one of the hallmarks of heterochromatin (Lister et al., 2009; Richards & Elgin, 2002;
Rountree & Selker, 2010; Sheaffer et al., 2014; Ziller et al., 2013, 2018), and has been
10
shown to restrict pluripotency in progenitor cells by preventing transcription factor
binding and permanently silencing genes (Domcke et al., 2015; Meissner et al., 2008;
Mohn et al., 2008). In the present study, we first characterized changes in DNA
methylation of supporting cells between birth and weaning at postnatal day 21. We
found that de novo CpG methylation encroaches on DNA methylation “valleys” of
developmentally regulated hair cell genes in supporting cells, and that this increase in
DNA methylation coincides with the loss of chromatin accessibility of hair cell loci in
supporting cells. This observation is the opposite of that of the intestinal epithelium,
where developmental enhancers retain a DNA demethylated state to allow for quick re-
activation as Lgr5+ intestinal stem cells proliferate and differentiate (Jadhav et al.,
2019). This difference in DNA methylation dynamics may explain why intestinal tissues
demonstrate a robust ability to regenerate throughout life, whereas the cochlear sensory
epithelium fails to regenerate during perinatal stages. This is also a rare example within
developmental biology describing the shutting down of developmental CpG island
promoter genes through active accumulation of DNA methylation. The more prominent
examples of this phenomenon occurs within the context of hypermethylation and
silencing of tumor suppressor genes in cancers (Esteller, 2002, 2007). To our
knowledge, we are reporting the first documented example of DNA hypermethylation to
restrict cell states in end stage differentiation trajectories.
We also found that supporting cells undergoing transdifferentiation induce de
novo DNA demethylation on a set of hair cell-specific regulatory elements. We further
demonstrated that inhibiting TET DNA demethylases prevents supporting cell
11
transdifferentiation in response to Notch inhibition, suggesting that young perinatal
supporting cells need to overcome DNA methylation barriers before they can
transdifferentiate into hair cells. This further suggests that upon DAPT treatment, Atoh1
de-repression and expression triggers the recruitment of TET enzymes to hair cell
genes. Whether Atoh1 is coordinating the recruitment of TET enzymes or this is
mediated through another protein, is unclear.
Lastly, we found that hair cell CpG island promoter genes continue to lose
chromatin accessibility at the single-cell level in every supporting cell subtype as they
reach adulthood. Interestingly, we found that supporting cells from mice that had been
deafened for long periods of time showed partial recovery of chromatin accessibility at
hair cell loci, suggesting these loci may be substrates for translational efforts to promote
hearing restoration. Most interestingly, we observe some Myo7a mRNA transcripts in
the P70 deafened border cell population that coincides with increased in chromatin
accessibility, suggesting that at some level, part of the hair cell gene program is
becoming de-repressed in the deafened state. This is promising for strategies to
regenerate hair cells from surviving supporting cells after hair cells have died off, and
suggests that border cells may be the most responsive target supporting cell sub-
population for future therapies in hearing restoration research. With this dataset, we
also identified both cell type-specific and stage-specific enhancers, such as the Gjb2
and Gjb6 enhancers, which are only accessible in P70 supporting cells.
Our work provides the first epigenetic analysis of heterochromatin formation in
the inner ear as the cochlea matures and loses its regenerative ability. Understanding
12
what barriers exist is critically important for future reprogramming studies which would
ultimately have to circumvent these repressive barriers.

Background and Significance
The leading cause of hearing loss is the damage and death of mechanosensory
hair cells (HCs), which can occur naturally with old age, through prolonged exposure to
loud environments, or by the use of ototoxic drugs (Groves, 2010). During the
development of the cochlear sensory epithelium, a subset of cells at embryonic day
13.5 (E13.5) upregulate p27Kip1 and become post-mitotic, giving rise to the prosensory
progenitor cell (PG) population (P. Chen & Segil, 1999). At E14.5, PGs broadly
upregulate the basic helix-loop-helix (bHLH) transcription factor(TF) Atoh1 to specify HC
fate (Bermingham et al., 1999). Subsequent Notch-mediated lateral inhibition in
adjacent cells downregulates Atoh1 to partition the supporting cell (SC) fate (P. Chen et
al., 2002). Throughout adulthood of non-mammalian species, HCs maintain the ability to
regenerate in response to trauma via the proliferation and transdifferentiation of
neighboring SCs (Lewis et al., 2012). The transdifferentiation process is similarly
marked by the upregulation of Atoh1. However, this is not the case in mammalian
species, where HCs rapidly lose their ability to regenerate postnatally (Kelley et al.,
1995).
In early perinatal mice, SCs maintain limited potential for transdifferentiation,
where either overexpression of Atoh1 or disruption of Notch signaling causes SCs to
convert to a HC-like state (Doetzlhofer et al., 2009; White et al., 2006; Zhao et al.,
13
2011). However, approaching the onset of hearing around postnatal day 12 (P12), SCs
quickly lose this transdifferentiation potential (Figure 1.5A, B) (Kelly et al., 2012; Maass
et al., 2015). Preliminary data from Z.P. Stojanova in our lab shows co-treating cochlear
explants with the Notch inhibitor DAPT and 3-deazaneplanocin A (DZNep) restores SC
transdifferentiation potential as shown by upregulation of the HC-specific genes Atoh1
and Myo6, and downregulation of the SC-specific gene Hes5 (Figure 1.5C). DZNep is a
nonspecific methyltransferase inhibitor, and it has been shown to improve induced
pluripotent stem cell (iPSC) reprogramming by decreasing DNA methylation and
promoting expression of Oct4 (Hou et al., 2013). Considering that DZNep inhibits S-
adenosylmethionine (SAM) synthesis, it could be perturbing any of the following histone
methylation marks: H3K27me3, H3K9me2, and H3K9me3 (Borchardt et al., 1984;
Miranda et al., 2009). Along these lines, preliminary data from L. Tao in our lab shows
that H3K27me3 increases in SCs from P1 to P6 (Figure 1.6B, C). Although H3K27me3
alone has the potential to silence genes (Boyer et al., 2006), interactions between
H3K27me3, H3K9me2, and H3K9me3, and their respective histone methyltransferases
have been suggested to cooperatively silence genes and spread heterochromatin
(Boros et al., 2014; Mozzetta et al., 2014). The methyltransferases G9a and G9a-like
protein (GLP) form heterodimers to establish H3K9me1 and H3K9me2 (Tachibana et
al., 2005), whereas Suv39h1, Suv39h2, and Setdb1 establish H3K9me2 and H3K9me3
(Peters et al., 2001; Rea et al., 2000; Schultz et al., 2002). DNA methylation is also
involved in this crosstalk, where DNA methyltransferases (DNMT) Dnmt3a and Dnmt3b
have been shown to be recruited by G9a and Suv39h1 to establish de novo DNA
14
methylation (Epsztejn-Litman et al., 2008; Lehnertz et al., 2003). Finally, feedback loops
between DNA methylation, H3K9 methylation, and H3K27 methylation recruit
heterochromatin protein 1 (HP1) to form heterochromatic regions (Boros et al., 2014;
Fuks et al., 2003). Based on our preliminary data regarding DZNep, it remains unclear
whether the individual methylation mark or their cumulative interplay during
heterochromatin formation is responsible for DZNep-induced Atoh1 upregulation in
maturing SCs.
I hypothesize that, along with H3K27me3, maturing SCs accumulate DNA
methylation (Aim 1) and H3K9 methylation (Aim 2) in supporting cells as part of the
heterochromatin maturation process. These marks would be responsible for repressing
hair cell-specific gene regulatory networks required for transdifferentiation. It is
important to consider the role of each mark independently because varying
combinations of each mark creates heterochromatin subtypes that may uniquely
regulate transcription (Becker et al., 2016). By studying the spread of heterochromatic
features in maturing SCs, I hope to understand how transdifferentiation potential is lost,
which will guide future endeavors to regenerate hair cells and restore hearing.

15

Figure 1.5 DNMT inhibitor RG108 increases percentage of supporting cell
transdifferentiation in response to DAPT treatment.
(A) Schematic of experiment paradigm. P0 cochlear explants from Atoh1-fGFP Lfng-
CreER TDT mice were treated with either (B) DMSO or (C) 4μm RG108 (DNMT
inhibitor) for 48hr followed by co-treatment with 10μM DAPT for 48hr.
Transdifferentiated SCs are double labeled (GFP+ TDT+). Pou4f3 labels total HCs. (D).
FACS quantification of transdifferentiation rate as the percentage of GFP+ TDT+ cells
over total TDT+ cells. (E) FACS quantified transdifferentiation rate of P0 explants pre-
treated with DMSO or RG108 followed by DAPT. Cochlear explants were split into
apical and basal halves. n=1 cochlea per condition. (E) Quantification of total Pou4f3+
HCs. (n=3, *p < 0.05, mean ± SEM).

16

Figure 1.6. DNMT inhibitor RG108 increases percentage of supporting cell
transdifferentiation in response to DAPT treatment.
(A) Example of H3K27me3 microChIP-seq peak at the Atoh1 locus (chr6:64,726,784-
64,738,246) in P6 SCs (B) Heatmap of H3K27me3 signal around transcription start sites
of HC-specific genes in SCs at E17, P1, and P6. (C) Average of heatmap signal in (B).
This data is courtesy of Litao Tao.

17
Chapter 2. NuTRAP reporter mice allow isolation of nuclei from
the postnatal cochlea for epigenetic profiling
Introduction
As the mouse organ of Corti matures during the first postnatal week, the ability of
supporting cells to transdifferentiate into hair cells is lost. We hypothesized that this
results from changes in the epigenetic status of the hair cell gene regulatory network
during maturation of supporting cells. To test this, we characterized the chromatin and
epigenetic profiles of maturing hair cells and supporting cells of the postnatal and adult
mouse cochlea. However, the maturing cochlea becomes increasingly difficult to isolate
with age, as the organ of Corti structure is solidified with tight junctions and extracellular
matrix (ECM) (Laine et al., 2007; Mustapha et al., 2009) (Figure 2.1). Furthermore, the
adult mouse cochlea microdissection is difficult and requires a high level of technical
expertise to isolate high quality cell types due to the calcification of the temporal bone
surrounding the inner ear epithelium.
To increase the yield of material from the adult mouse cochlea, we used the
NuTRAP mouse labeling system in combination with mechanical and detergent-based
homogenization of adult cochlear tissue to release intact, high-quality nuclei for
downstream NGS applications such as gene expression and epigenetic profiling.

18

Figure 2.1 Morphological changes of organ of Corti during postnatal maturation.
Cross sections of the cochlea reveal organ of Corti subunits at P3, P6, P10, and P21.
Morphological changes are observed as the organ of Corti matures and transitions from
an epithelial tissue to a more specialized structure for hearing detection.

19
Mechanical and detergent-based lysis of cochlea epithelia releases high
quality nuclei for downstream NGS applications
Previous methods of obtaining single cell suspensions from the mouse cochlea
involve the use of enzymes such as trypsin, dispase, and collagenase to digest the
proteins adhering the cell membranes to each other. However, as the cochlea sensory
epithelia matures, tight junctions and cytoskeleton are established to solidify the
structure of the organ of Corti as it is preparing for hearing functionality. This makes the
cochlea sensory epithelia refractory to traditional enzymes. Additionally, using other
enzymes such as thermolysin to enable on-ice digestion and preserve mRNA quality
and chromatin structure makes the digestion time too long and is still ineffective to
recover high number of cells. Papain, derived from papayas, has strong digestive
properties, but proved to be too harsh and completely digests and lyses the epithelia
without any cell recovery. Other single cell transcriptomic profiling studies within the
inner ear that use enzymatic digestion of adult cochlear tissue followed by pipet flushing
of the cochlear duct have reported on the enrichment of entirely cell populations derived
from the stria vascularis and not the cochlear sensory epithelia (Korrapati et al., 2019).
This suggests the failure to dissociate and recover the actual target cochlear cell types
using current enzymatic methods. Many labs avoid ineffective enzymatic digestion
completely in favor of lysing the unsorted cochlear tissue itself as input for next
generation sequencing epigenetic profiling experiments (Yizhar-Barnea et al., 2018).
These experiments are not informative as they grossly simplify the cochlear tissue, and
do not address the complex heterogeneity and contributions from highly specialized and
distinct cell types within the cochlea.
20
Given these preliminary studies in both our lab and in the inner ear field, we
decided to move away from enzyme-based digestion methods in favor of mechanical
and detergent-based lysis to break cell membranes to release intact nuclei. This method
has not been described within the ear field to date but is commonly used for the
extraction of nuclei from brain tissue. The protocol used in this study has been
optimized from previously protocols developed for the brain (Casals & Mitsios, 2019;
Nott et al., 2021) with modifications such as skipping the myelin removal step that is
specific to brain tissue, but the main concepts are retained. The homogenization buffer
was formulated to optimize nuclear recovery (Table 3.1). The addition of sucrose and
glycerol, two high viscosity agents was to help buffer and stabilize the nuclear
membrane during mechanical homogenization. KCl and MgCl2 were added to balance
tonicity. MgCl2 is also required to stabilize nuclear envelope proteins and overall nuclei
integrity during extraction. The detergent NP-40 was added at 0.1% final concentration
to facilitate the breaking of cell membranes during homogenization.
Cochlear tissues were then dounce homogenized in the homogenization buffer in
a 1ml dounce homogenizer. Six to twenty cochlear tissues can be processed in a 1ml
dounce homogenizer taking great care to remove bone debris and excessive tissue to
improve the quality of released nuclei during dounce homogenization. Excess off-target
tissue can overrepresent the target cell population and See Materials and Methods for
more details. Briefly, cochlear tissues were micro-dissected from the cochlea and
placed in a modified artificial cerebral spinal fluid (ACSF) solution in a dounce
homogenizer chilled on ice. The ACSF solution was formulated with salts to mimic the
21
endocochlear potential that the hair cells and supporting cells interface within the
cochlea (Table 3.2). Once all cochlear tissue was harvested, mechanical
homogenization was performed to break up the tissue and release nuclei: quick up
strokes were followed by slow down strokes, taking care to avoid bubbles introduced by
lifting the pestle above the solution layer. Post homogenization lysates were filtered to
remove debris. Depending on the sensitivity of downstream applications, we employed
either a gradient separation method or a 10 micron filter to separate released nuclei
from extracellular debris (Figure 3.2). In either case, 2 ml of NEB was added to dilute
the homogenization buffer prior to spinning down nuclei into a pellet at 500 x g for 10
minutes at 4°C in a swing bucket centrifuge. Nuclei were resuspended in nuclear
extraction buffer (NEB) (Table 3.3) and ready for fluorescent activated cell sorting
(FACS)-mediated purification. Great care was taken to remove the high viscosity
glycerol and sucrose solution from the nuclear pellet through washing with NEB, to
prevent droplet clogging in the FACS.
Nuclear quality was assessed after each step of the protocol and adjustments
were made to optimize nuclear quality for a particular tissue or sample type. Common
parameters requiring empirical determinations included: the BSA concentration, range
0.1% to 2% in homogenization buffer, ACSF, or NEB; the amount of NP-40, range from
0.1%-0.5% in the homogenization buffer and NEB; and the number of strokes with loose
and tight pestles, up to 20 strokes, but users should aim for partial lysis rather than
complete lysis of tissues, as the majority of nuclei can be released regardless of
remaining intact tissues. Over-homogenization can lyse nuclei and released DNA
22
induces excessive clumping. The use of an iodixanol density gradient improves nuclear
quality but extends the protocol duration and incurs transfer loss. If the downstream
application does not require such high-quality nuclei, users can opt for the 10 micron
cell strainer to filter nuclei from cell debris. The optimized buffers described in this
workflow already ensures for high quality nuclei recovery, and we find that the iodixanol
gradient yields marginal improvements to nuclei quality with too high a tradeoff in time.
In conclusion, this protocol yields high quality nuclei from tough-to-dissociate
adult cochlear tissue and is suitable for downstream next-generation sequencing (NGS)
experiments such as transcriptomics and epigenetic profiling. This protocol also
improves upon previous enzyme-based methods of dissociating cochlear epithelia at
adult stages and extends the window of which we can isolate hair cells and supporting
cells up to at least P21 for hair cells and P70 for supporting cells (Figure 3.3).

23
Table 2.1. Homogenization Buffer
Homogenization Buffer
Stable for 6 months at 4°C
Amount (50 ml)
0.25 M Sucrose (8.5%)
5ml of 2.5M sucrose solution
25mM KCl
625ul of 2M stock
5 mM MgCl2
250ul of 1M stock
20% glycerol
12.5ml of 80% Glycerol
20 mM HEPES-KOH, pH 7.9
1ml of 1M HEPES
dH2O
30.625 ml
Add the following components before use
Make 4 ml; requires 2 ml / sample
1mM DTT 4ul of 1M stock
0.15 mM Spermine 0.6 ul of 1M stock
0.5 mM Spermidine 1 ul of 2M stock
2% BSA, fraction V 266 ml of 30% BSA, fraction V
cOmplete EDTA-free Protease inhibitor
cocktail tablet
1x

Table 2.2. Artificial Cerebral Spinal Fluid (ACSF)
ACSF mM
NaCl 119
KCl 2.5
CaCl2 2.5
MgCl2 1.3
NaH2PO4 1
NaHCO3 26.2
D-Glucose 11

24
Figure 2.2. Nuclei separation from cell debris.
After dounce homogenization, nuclei fraction are enriched using either (A) an iodixanol
density gradient causing nuclei to band at the 30%-40% density interface or (B) a 10
micron filter.

Table 2.3 Nuclear extraction buffer (NEB)
Nuclear extraction buffer pH 7.9 Amount (50 ml)
20 mM HEPES-KOH, pH 7.9 1ml of 1M HEPES
10mM KCl 250ul of 2M stock
0.1% NP-40 250ul of 20% NP-40
dH2O 48.5ml
Add the following components before use
Make 4 ml; requires 2 ml / sample
1mM DTT 4ul of 1M stock (-20C, Litao’s box)

0.15 mM Spermine 0.6 ul of 1M stock (-20C, side door)
0.5 mM Spermidine 1 ul of 2M stock (-20C, blue box)
2% BSA, fraction V 266ml of 30% BSA
cOmplete
EDTA-free Protease inhibitor cocktail tablet
(Roche 11836170001, lot:39802300)
1x

25

Figure 2.3.Enzyme-based dissociation versus dounce homogenization of cochlear
tissue.
Comparison of enzymatic digestion using a cocktail of trypsin, dispase, and collagenase
versus mechanical and detergent-based Dounce homogenization lysis from cochlear
tissue at P1, P6, P21, and P70 stages. Cell and nuclei recovery are reported for hair
cells (HC) and supporting cells (SC) at each time point.

26
Using the NuTRAP transgenic mouse to label nuclei of target cell
populations
To increase the yield of material from the adult mouse cochlea, we used the
NuTRAP mouse labeling system (Roh et al., 2017) (Figure 3.4). Other methods of
trangenically labeling nuclei include the SunTag system, which localizes antibody-fused
to a GFP fluorescent protein to a novel protein scaffold consisting of a peptide array
termed SunTag (Tanenbaum et al., 2014). This peptide recruits up to 24 copies of GFP
via antibody interactions and allows for live imaging in cells. Other strategies use a cell
type-specific nuclear marker, which is formaldehyde cross-linked, and tagged with
fluorescent antibodies to the cell type-specific nuclear marker for FACS purification
(Bonn et al., 2012). However, the limitation of this method is that there must be a known
cell type-specific nuclear membrane marker. Another method is to selectively tag nuclei
via the H2B-GFP fusion protein (Jiang et al., 2008). However, the caveat here is that the
GFP fused to H2B may also induce some chromatin changes that are not
representative of naïve biological state. Others have tried fusing a BLRP biotin
substrate to a nuclear localization marker such as Ran-GAP1 to tether BLRP to the
nuclear envelope (Deal & Henikoff, 2010). BLRP is the substrate for BirA biotinylating.
The caveat with this method is that there might not be any biotin available within the
tissue in question, making the reaction non-reactive. The opposing condition can also
be true, where the system is too abundant in biotin, which if not adequately removed,
the free biotin will compete with streptavidin binding and block enrichment of the target
nuclei population.
27
Following cell type-specific Cre-mediated recombination, the NuTRAP mouse
labels the nuclear membrane with mCherry, and the ribosomes with GFP. Atoh1 is one
of the earliest markers of differentiating hair cells, and Lunatic Fringe (Lfng) is
expressed in most supporting cells in the postnatal cochlea. We crossed Atoh1-CreER
or Lfng-CreER mice with NuTRAP reporter mice to label hair cells and supporting cells,
respectively. An example of Atoh1-CreER NuTRAP labeling is shown (Figure 3.5).
Under this system, we see robust labeling of nuclei, the majority of hair cells were
labeled, recapitulating the pattern of 3 rows of outer hair cells and 1 row of inner hair
cells. Atoh1-creER also drives NuTRAP labeling of both type I and type II hair cells
within the utricle (Figure 3.5).
Initially, we tried to enrich for the NuTRAP-labeled nuclei using the biotinylation
of BLRP with streptavidin-magnetic beads as the authors of the NuTRAP mouse
intended. However, this approach did not sufficiently exclude non-specific nuclei from
our target population. Further, streptavidin-magnetic beads induced excessive nuclear
clumping, which after filtration, significantly reduced the number of nuclei recovered in
our positive-selected fraction. Therefore, we opted to move to FACS-mediated
purification of nuclei. Briefly, dissected cochlear tissue was processed with a Dounce
homogenizer to release nuclei yielding high quality NuTRAP-labeled nuclei with minimal
debris (Figure 3.6). Nuclei were then FACS-purified to isolate labeled nuclei of interest
(Figure 3.7). Initially, FACS-sorting was performed on the mCherry signal associated
with the nuclear membrane. However, this mCherry signal bleached rapidly and the
GFP signal associated with ribosomes in the nucleolus proved to be robust and stable
28
(Figure 3.7). The EGFP-L10a fluorescent signal was particularly robust inside the
nucleolus and was maintained throughout the extraction process. NuTRAP-labeled
nuclei were then FACS purified based on the EGFP signal. This protocol was suitable
for downstream epigenetic profiling experiments such as CUT&RUN, CUT&TAG, whole
genome bisulfite sequencing (WGBS), and single cell multiomics.

29

Figure 2.4. NuTRAP transgenic mouse labeling system of nuclei
After Cre-mediated removal of a loxP-flanked Stop cassette, the NuTRAP mouse labels
the nuclear envelope with the fluorescent protein mCherry, while labeling ribosomes
with GFP. mCherry is tethered to RanGAP1, a nuclear localization signal protein, and to
BLRP which is the substrate for biotinylation by BirA enzyme. EGFP fluorescent protein
is fused to the L10a ribosomal subunit. 2A self-cleaving peptides are inserted to
separate proteins within the transgene.

30

Figure 2.5. Atoh1-creER NuTRAP mouse labels hair cells of the cochlea and utricle
Atoh1-CreER drives NuTRAP expression in the hair cells of the cochlea and utricle.
mCherry labels the nuclear envelope, while GFP labels ribosomes in the nucleolus.
Scale bars for cochlea and utricle are 20µm and 100µm, respectively.

Figure 2.6. NuTRAP nuclei after dounce homogenization and iodixanol gradient
separation.
(A) Two NuTRAP labeled nuclei can be spotted amongst unlabeled nuclei. (B) 250x
digital zoom of released nuclei shows no blebbing and is indicative of preserved nuclear
membrane integrity.
31

Figure 2.7. FACS purification of NuTRAP labeled nuclei
(A) NuTRAP-labeled nuclei are FACS-purified on a BD Aria I. (B) NuTRAP-labeled
nuclei are FACS-purified from the GFP-tagged ribosomes located in the nucleolus
puncta. (C) Approximately 5.1% of the total nuclei population are enriched for Lfng-
creER NuTRAP-labeled supporting cells from a P6 mouse.

32
Chapter 3. Epigenetic maturation of organ of Corti supporting
cells and the hair cell gene regulatory network
Introduction
During cochlear development, the regulated expression of the bHLH E-box
transcription factor Atoh1 is required for proper differentiation of the mechanosensory
hair cell population. Previous studies from the Segil Lab demonstrate dynamic changes
in H3K4me3, H3K27me3, H3K9ac, and H3K9me3 at the Atoh1 locus, suggesting a
transition from a poised, to active, and finally, to a repressed epigenetic state which also
coincides with the initial upregulation of Atoh1 and its eventual downregulation and
silencing by postnatal timepoints (Stojanova et al., 2015). Furthermore, this tight
temporal regulation of Atoh1 expression may be regulated by Pou4f3 pioneer factor
activity, where Atoh1 initial expression targets the downstream expression of the
pioneering transcription factor, Pou4f3. Pou4f3 is then required to bind to closed Atoh1
binding sites and provide access for Atoh1 binding (H. V. Yu et al., 2021). This feed-
forward mechanism between Atoh1 and Pou4f3 is required for activation of the
complete hair cell gene regulatory network and is required for continued differentiation
of hair cells. After hair cell gene regulatory network activation, supporting cells undergo
a wave of enhancer decommissioning of hair cell-specific enhancers, where hair cell
enhancers lose H3K4me1 priming within the first postnatal week and prevents the
readily activation of Atoh1 target genes (Tao et al., 2021). These initial studies inform us
on how the hair cell gene regulatory network is first established in the prosensory Sox2+
progenitor population, and its subsequent turning off by P6 which may explain why
supporting cells fail to respond to DAPT by P6. However, it is still unknown whether
33
additional epigenetic changes occur after P6 to further silence the hair cell gene
regulatory network in supporting cells. This knowledge will be critical because it more
closely resembles the adult epigenetic landscape. Therefore, we set out to characterize
epigenetic changes at these later postnatal timepoints up to P21 using the NuTRAP
nuclei labeling and isolation protocol.
Cochlear progenitor cells, hair cells and supporting cells have distinct DNA
methylation signatures.
We hypothesized that systemic heterochromatinization contributes to the loss of
transdifferentiation potential in supporting cells. To identify genomic regions undergoing
heterochromatinization in maturing postnatal supporting cells, we examined DNA
methylation patterns. We profiled DNA methylation using WGBS on cells from the organ
of Corti at the following key developmental stages: embryonic day 13.5 prosensory
progenitor cells (E13.5 PG), postnatal day 1 hair cells (P1 HC), postnatal day 1
supporting cells (P1 SC), postnatal day 6 supporting cells (P6 SC), and postnatal day
21 supporting cells (P21 SC). We isolated E13.5 prosensory progenitors from p27Kip1-
GFP mice, P1 hair cells from Atoh1-GFP mice, and P1 supporting cells from Lfng-EGFP
mice. For P6 and P21 supporting cells, we used the NuTRAP method described above
to isolate nuclei from Lfng-CreER; NuTRAP mice. As a positive control, we confirmed
that DNA methylation is depleted at 13,564 CGI promoters and enriched 8,586 non-CGI
promoters (Figure 3.1A).
We observed distinct methylomes in the different cell types and time points. We
observed that the Atoh1 and Dll1 loci are more heavily methylated in P21 supporting
34

Figure 3.1. Quality control analysis of WGBS datasets depicts typical characteristics of
mCpG distribution
(A) Proportion of total identified DMRs overlapping with CGI promoter versus non-CGI
promoters. (B) ATOH1 and POU4F3 motifs enriched in de novo HC-specific DMRs. (C)
phastCons 60-way conservation scores were plotted as average profiles at each DMR.
(D) mCpG percentage at CGI promoters and non-CGI promoters as positive control for
identified DMRs. H3K4me3 and H3K4me1 CUT&Tag signal are displayed to visualize
promoter center. Promoter regions are centered on called H3K4me3 peaks with a ±5 kb
window. (E) mCpG percentage over cochlea-specific DMRs in intestinal Lgr5+ ISCs and
enterocytes as negative control for identified DMRs.

35
cells compared to E13.5 prosensory progenitors, P1 hair cells, P1 supporting cells, and
P6 supporting cells (Figure 3.2A). Since the ATOH1 transcription factor is both
necessary and sufficient for hair cell differentiation, we profiled ATOH1 chromatin
binding regions in E17.5 hair cells. We used CUT&RUN with anti-GFP antibodies to
identify binding sites in hair cells obtained from Atoh1-EGFP mice. We found ATOH1
binding sites at both the promoter and enhancers of the Atoh1 locus, including a
previously characterized 3’ autoregulatory enhancer overlap with regions of DNA
hypermethylation in P21 supporting cells (Figure 3.2A). We saw a similar pattern in the
Notch ligand Dll1, which is specifically expressed in hair cells (Figure 3.2A). Since
ATOH1 and DLL1 play critical roles in hair cell and supporting cell fate specification,

36

37
Figure 3.2. Distinct DNA methylation patterns are enriched at select hair cell and
supporting cell enhancers
(A) Genome track showing the Atoh1 locus with ATOH1 protein binding measured by
CUT&RUN. DNA methylation tracks of E13.5 progenitors (PG), P1 hair cells (HC), P1
supporting cells (SC), P6 SC, and P21 SC show gradual accumulation of methylated
CpGs within the DNA methylation valley of the Atoh1 locus as supporting cells mature
from P1 to P21. Methylation state of CpGs displayed as percentage, where full line
represents 100% methylation and absence of a line represents 0% methylation. CpG
islands, shores, and shelves are annotated. (B) PCA analysis of CpG methylation
percentage separated by CpG islands, promoters, and enhancers. (C) Differentially
methylated regions (DMRs) between cell types and developmental stages were
identified using methylKit. Four categories of DMRs were identified: pre-established
regions were hypomethylated in the E13.5 PG stage and later hypermethylated in P21
SC; de novo common regions were hypermethylated in E13.5 PGs and only
hypomethylated in HCs and SCs; de novo HC-specific regions were only
hypomethylated in P1 HCs; de novo SC-specific regions were gradually hypomethylated
in SCs between P1 and P21. Blue corresponds to 100% methylation, whereas red
corresponds to 0% methylation. DMRs centered on ATOH1 peaks with a ±5 kb window.
(D) ATOH1 and POU4F3 CUT&RUN binding in E17 HC, as well as H3K4me3 and
H3K4me1 CUT&Tag signal plotted over DMRs. (E) Gene ontology (GO) terms of genes
associated with each respective DMR.

38
DNA hypermethylation of these gene loci suggests a larger genome-wide shutdown of
gene programs responsible for the development of hair cells and supporting cells with
increasing age.
We characterized the changes in DNA methylation on a genome-wide level in our
samples using principal component analysis (PCA), seoarating DNA methylation data
into different genomic features - CpG islands, promoters, and enhancers - prior to PCA
analysis (Figure 3.2B). At CpG islands, E13.5 prosensory progenitors, P1 hair cells, and
P1 supporting cells clustered together, while P6 supporting cells and P21 supporting
cells clustered away from E13.5 prosensory progenitors, P1 hair cells, P1 supporting
cells, and from each other (Figure 3.2B, left). This clustering pattern suggests that DNA
methylation at CpG islands stays relatively constant among cell types (prosensory
progenitors, hair cells, and supporting cells) at earlier stages of development, but
diverges by the end of the first postnatal week. At gene promoters, all
cell types and stages clustered separately, with E13.5 prosensory progenitors in the
middle and hair cells and supporting cells diverging towards separate identities (Figure
3.2B, middle). This suggests that there are distinguishing DNA methylation patterns at
promoters between specific cell types and stages of development in the organ of Corti.
At gene enhancers, P1 hair cells and P1 supporting cells cluster away from the
prosensory progenitor state but are relatively similar to each other, which might suggest
that they still share similar enhancer networks at this earlier stage of development,
consistent with our previous data (Figure 3.2B, right) (Tao et al., 2021). Our data
39
suggest that DNA methylation is positively correlated with the increasing maturation of
supporting cells with age.
Next, we sought to identify the genomic regions driving the variance between the
cell types that could contribute to distinct gene silencing patterns. We used dmrseq to
identify differentially methylated regions (DMRs) between prosensory progenitors, hair
cells, and supporting cells of different ages to obtain region sets that were
hypermethylated or hypomethylated in one group versus the other. We further
intersected the DMR sets to obtain four mutually exclusive region sets, which we named
according to the dynamics of their DNA methylation signature (Figure 3.2C). All four
sets of DMRs showed evolutionary conservation, suggesting that they possess some
regulatory function and giving confidence to the peak calling method (Figure 3.1C). We
identified 2,850 pre-established DMRs, characterized by being hypomethylated in E13.5
prosensory progenitors, P1 hair cells and P1 supporting cells, and becoming
progressively hypermethylated in P6 and P21 supporting cells. We identified 6,746 de
novo hair cell-specific DMRs, which are uniquely hypomethylated in P1 hair cells
and3,058 de novo supporting cell-specific DMRs, which become most heavily
hypomethylated in P21 supporting cells. Finally, we identified 9,683 de novo common
DMRs, which are hypomethylated in P1 hair cells, P1 supporting cells, P6 supporting
cells, and P21 supporting cells (Figure 3.2C). Both the pre-established and de novo hair
cell-specific DMRs showed enrichment of the ATOH1 E-Box and POU4F3 homeobox
motifs, suggesting these DMRs are part of the hair cell gene regulatory network (Figure
3.1B). Strikingly, the DMRs appear to be highly specific to organ of Corti cells, as the
40
DMRs are completely hypermethylated in intestinal tissues, even though both tissues
express ATOH1 and regulate cell fate by Notch signaling (Figure 3.1E) (Durand et al.,
2012; Jadhav et al., 2019; Stanger et al., 2005). The de novo distinction we used is
important, because the prosensory progenitors, hair cells, and supporting cells are
postmitotic at all developmental stages examined in this study. Thus, the mechanism of
DNA hypomethylation is most likely mediated by TET enzyme activity, rather than by
passive DNA demethylation during cell division (Monk et al., 1991; Tahiliani et al., 2009;
F. Zhang et al., 2007). Taken together, our analysis shows that DNA methylation
patterns coincide with distinct developmental stages and cell types.

DNA methylation signatures highlight gene regulatory network switching
within the prosensory epithelium as it matures.
To determine if the DMRs identified in our analysis were part of the hair cell gene
regulatory network, we overlaid the DMR sets with binding sites for two key hair cell
transcription factors, ATOH1 and POU4F3. POU4F3 is a downstream target of ATOH1
and has pioneer transcription factor activity, accessing closed ATOH1 target enhancers
and allowing hair cell differentiation to proceed (H. V. Yu et al., 2021). We used
published ATOH1 and POU4F3 CUT&RUN data from FACS-purified E17 hair cells (Tao
et al., 2021; H. V. Yu et al., 2021) (Figure 3.2D). The de novo hair cell-specific DMRs
and the de novo common DMRs contain both ATOH1 and POU4F3 binding sites,
suggesting they might be downstream targets of ATOH1 and POU4F3, and are
therefore required for the differentiation of hair cells. On the other hand, the pre-
established DMRs and the de novo supporting cell-specific DMRs are only bound by
41
ATOH1, and not by POU4F3 (Figure 3.2D). Since the pre-established DMRs are
hypomethylated in E13.5 prosensory progenitors, we suggest these regions may serve
as initial targets of ATOH1, which are upregulated within the prosensory domain at
E14.5 to initiate hair cell differentiation (P. Chen et al., 2002; H. V. Yu et al., 2021). To
further categorize these DMR sets, we performed CUT&TAG on the promoter-specific
mark H3K4me3 and the promoter-enhancer mark H3K4me1 in P1 hair cells and P1
supporting cells (Kaya-Okur et al., 2019). DMR sets are not enriched for H3K4me3 but
are enriched for H3K4me1 in both hair cells and supporting cells (Figure 3.2D),
suggesting that the DMRs are hair cell- and supporting cell-specific enhancers.
Pre-established DMRs include known organ of Corti developmental genes such
as Sox2, Atoh1, Gfi1, Notch1, and Dll1. Gene ontology (GO) enrichment analysis on the
DMR sets using GREAT (C. Y. McLean et al., 2010). showed pre-established DMRs are
enriched for GO terms relating to “regulation of epithelial cell differentiation”, “stem cell
population maintenance”, and “regulation of auditory receptor cell differentiation” (Figure
3.2E). The de novo hair cell-specific DMRs are enriched for terms relating to the
maturation of hair cells, such as “neuromuscular process controlling balance”,
“mitochondrial membrane organization”, and “auditory receptor cell stereocilium
organization”. Genes within the de novo hair cell-specific DMRs include early hair cell
developmental genes like Atoh1, Pou4f3, Gfi1, Myo7a, Myo6, and Jag2, as well as
genes involved in the maturation of hair cells such as Pcdh15, Cdh23, and Clic5. The
de novo supporting cell-specific DMRs have GO terms relating to epithelium formation
with terms such as “cell-cell junction organization”, and “positive regulation of
42
cytoskeleton organization.” Key genes include Sox2, Sox4, Sox9, Gjb2, Gjb6, Coch,
KCN (potassium channel), and SLC (solute carrier) family genes. Finally, the de novo
common DMRs have GO terms relating to more general epithelial maturation, such as
“cytoskeleton-dependent intracellular transport” and “adherens junction organization”
and broadly include KCN, SLC, and CDH (cadherin) family genes. The GO analyses
demonstrates that each organ of Corti cell type has a distinct DNA methylation
signature at each age that correlates with its known gene expression and function. We
suggest that dynamic DNA methylation patterns are used to reinforce gene program
switches as cells transition through different states and identities]. For instance, as
supporting cells lose the ability to transdifferentiate postnatally, we observed a switch
from a hair cell differentiation gene program to a supporting cell maturation gene
program, where the former becomes hypermethylated and the latter becomes
hypomethylated.

DNA methylation encroachment and heterochromatinization of pre-
established CpG island promoters occurs in postnatally maturing
supporting cells.
The gradual DNA hypermethylation of pre-established DMRs in postnatal
supporting cells over the first postnatal week correlates with the period during which
supporting cells lose the potential to transdifferentiate in response to DAPT (Lanford et
al., 1999; Yamamoto et al., 2006). Initially, the percentage of methylated CpGs at pre-
established DMRs decreased from 45% in E13.5 prosensory progenitors to 30% in P1
hair cells and P1 supporting cells, which coincided with the start of differentiation of
43
cochlear hair cells and supporting cells. The CpG percent methylation then increased
gradually to 38% and 50% by P6 and P21 in supporting cells, coinciding with loss of
transdifferentiation potential (Figure 3.3A). To better understand the role of DNA
hypermethylation in supporting cells, we annotated the pre-established DMRs to
genomic features (Cavalcante & Sartor, 2017), and found that they are enriched around
CpG islands (CGIs) and their respective shores and shelves (Figure 3.3B, C). CGIs are
genomic regions of 500 to 1,500bp long with a ratio of CpG dinucleotide sequences
greater than 0.6, and they are often found at promoters and retrotransposable elements
(Cross & Bird, 1995). In agreement with this, the pre-established DMRs are also
enriched around genic regions, spanning from 5kb upstream of the promoter, through
the gene body, and all the way to the 3’ UTR (Figure 3.3C). The pre-established DMRs
are not enriched at intergenic regions, suggesting that they are unlikely
retrotransposons or distal enhancers. Together, this indicates that the majority of pre-
established DMRs are enriched at CpG island-containing promoters.
CGI promoter genes serve important roles as transcriptional hubs and tend to be
developmentally regulated. They sit within DNA methylation valleys, also referred to as
DNA methylation canyons, which are largely hypomethylated (Xie et al., 2013). These
CGI promoter genes are frequently silenced by the repressive histone modification
H3K27me3 (Xie et al., 2013). The recruitment of PRC2, a H3K27me3-specific
methyltransferase, to CG rich sequences at promoters also repels DNA
methyltransferases (DNMTs) (Boyer et al., 2006; J.-S. Lee et al., 2010). Thus, CGI
promoters, used for lineage specification during the process of cell differentiation, are
44
maintained in a DNA hypomethylated state in differentiated cells. The current model of
CGI promoter repression is proposed to be mediated by H3K27me3 for both the pre-
lineage commitment bivalent state, as well as the subsequent repression of non-lineage
genes (Bernstein et al., 2006). DNA hypermethylation has been shown to occur at
promoters that are CG-poor and lack CGIs, which tend to be somatic tissue-specific
genes expressed in more mature cell types (Xie et al., 2013; Zhu et al., 2008). Building
on this model, we found that cochlear supporting cells undergo a switch between the
two modes of repression - H3K27me3 and DNA methylation - at developmentally
regulated hair cell-specific CGI promoter genes (Figure 3.3D). For example, at three key
hair cell-specific transcription factor gene loci, Atoh1, Pou4f3, and Gfi1, we find
evidence of both DNA hypermethylation and heterochromatinization. Additional
examples of DNA hypermethylation were observed at Tead4, Eya1, Notch1, and Fgfr3
(Figure 3.4A). On the genic level, the DNA hypermethylation of pre-established DMRs
manifests itself as DNA methylation encroachment of the DNA methylation valleys in
which the respective CGI promoter gene sits (Figure 3.3D, WBGS). These genes were
bivalent prior to lineage commitment but resolved to a repressive H3K27me3 state upon
loss of transdifferentiation potential. A clear example can be seen at the Pou4f3 locus,
where the promoter is marked by both the repressive histone modification H3K27me3,
and the permissive histone modification H3K4me1 in P1 hair cells and P1 supporting
cells, albeit at lower levels (Figure 3.3D). By P6, when the transdifferentiation potential
is gone, there is a complete depletion of H3K4me1.

45

46
Figure 3.3. Pre-established CGI promoters exhibit DNA methylation encroachment and
a silenced chromatin state in maturing supporting cells
(A) Average mCpG percentage in different cochelar cell types was plotted as average
profiles at pre-established DMRs (B) Distance of pre-established DMRs to nearest CpG
island is represented as a density plot. The y-axis measures the proportion of pre-
established DMRs within 10kb bins (C) Enrichment of pre-established DMRs over
annotated features in the genome. Enrichment score was calculated as log2 of
observed pre-established DMRs at each annotation type against regions obtained from
a random sampling of the genome. (D) Representative genomic tracks of hair cell-
specific genes Atoh1, Pou4f3, and Gfi1 loci overlaid with ATOH1 CUT&RUN signal.
Shows changes in percent mCpG, as well as H3K27me3, H3K4me1, and CUTAC
chromatin accessibility CUT&TAG signal between P1 HC, P1 SC, P6 SC, and P21 SC.
(E-H) The 2,850 pre-established DMRs overlapped with 103 CGI promoters.
Normalized read counts of (E) H3K27me3, (F) % mCpG, (G) CUTAC, and (H)
H3K4me1 were averaged over a 1kb window at the 103 promoters within each cell type
and stage (quartile distribution box plots).

47
Next, we identified the epigenetic changes occurring across 103 gene promoters
that overlapped with the previously identified set of 2,850 pre-established DMRs (Figure
3.3E-H). We observed a switch from H3K27me3-mediated repression to a DNA
methylation-mediated silencing in supporting cells between P6 and P21 (Figure 3.3E,
F). This finding is in accordance with previous work describing the antagonistic
relationship between DNA methylation and PRC2-mediated H3K27me3 deposition (Z.
Chen et al., 2019; Reddington et al., 2013). The switch from H3K27me3-mediated
repression to DNA methylation-mediated silencing was also observed at both HoxA and
HoxB loci (Figure 3.4B). We propose the switching between H3K27me3 and DNA
methylation is a mechanism for the long-term silencing of hair cell-specific CGI promoter
genes and the complete shutting down of the hair cell gene regulatory network (GRN).
As a positive control, we confirmed that H3K27me3 signal at promoters is comparable
across samples (Figure 3.4A).
In addition to DNA hypermethylation, DNA accessibility measured by Cleavage
Under Targeted Accessible Chromatin (CUTAC) (Henikoff et al., 2020) drops
precipitously between P1 and P21 in supporting cells (Figure 3.3G). While the increase
of H3K27me3 between P1 and P8 explains the decreased accessibility within that same
time window, DNA hypermethylation explains the decrease in accessibility between P8
and P21, suggesting that DNA hypermethylation is required in the final stages of
maturation of supporting cells to complete heterochromatinization of their hair cell-
specific CGI promoters. Finally, we detected a decrease in H3K4me1 signal over these
gene loci by P21 (Figure 3.3H), which provides further support for a silenced chromatin
48
state at these 103 gene loci, in this case by decommissioning of hair cell enhancers
(Tao et al., 2021). Besides CGI promoters, we also see similar changes in DNA
methylation, H3K27me3, H3K4me1, and accessibility at pre-established DMRs that
overlap with non-CGI promoters, gene bodies, and intergenic regions, albeit less
prominently (Figure 3.5B).

49

Figure 3.4. H3K27me3 signal is comparable across samples and is inversely correlated
to DNA methylation.
(A) Heatmap of H3K27me3 CUT&RUN signal at promoters shows comparable signal
intensity across cell types and stages. k-means clustering with k=2. Promoter regions
are centered on H3K4me3 called peaks with a ±5 kb window. (B) DNA methylation and
H3K27me3 signal are inversely correlated at Hox A and Hox B loci.

50

Figure 3.5. Characterization of H3K27me3, H3K4me1, and CUTAC signal at genomic
features.
(A) Representative genome tracks of key genes involved in cochlear sensory epithelium
development: Tead4, Eya1, Notch1, Fgfr3. Changes in ATOH1 CUT&RUN, percent
mCpG, as well as CUT&TAG signal of H3K27me3, H3K4me1, CUTAC are shown
between E13.5 PG, P1 HC, P1 SC, P6 SC, and P21 SC. (B) The 2,850 pre-established
DMRs overlapped with 744 non-CGI promoter regions, 11,587 gene body regions, and
1,582 intergenic regions. Normalized read counts of CUTAC, H3K4me1, H3K27me3, %
mCpG were averaged over a 1kb window at each set of regions (quartile distribution
box plots).
51
Chapter 4. DNA methylation in the mouse cochlea contributes to
the failure of hair cell regeneration
Introduction
Neonatal supporting cells still retain the potential to transdifferentiate into hair
cells after hair cell loss, or by blocking Notch signaling with the gamma-secretase
inhibitor DAPT (Cox et al., 2014; Doetzlhofer et al., 2009; Korrapati et al., 2013; Lanford
et al., 1999; Maass et al., 2015; Takebayashi et al., 2007; Yamamoto et al., 2006).
However, from our DNA methylation data, we observe that the de novo hair cell-specific
DMRs are hypermethylated in P1 supporting cells (Figure 3.2C). Based on the GO
terms previously described, the hair cell-specific DMRs are essential for hair cell
differentiation (Figure 3.2E). Since DNA methylation is a repressive epigenetic feature,
we hypothesized that in cases where P1 supporting cells differentiate into hair cells,
hypermethylated hair cell-specific DMRs in P1 supporting cells must first undergo
demethylation and derepression for supporting cells to successfully up-regulate hair
cell-specific genes during transdifferentiation.
DAPT-mediated transdifferentiation induces de novo DNA demethylation of
hair cell-specific DMRs
To test this, we induced P0 supporting cell transdifferentiation with DAPT and
performed WGBS to measure mCpG percentages in DAPT-responsive supporting cells
compared to DAPT-unresponsive cells. Briefly, we used a transgenic mouse line with
Lfng-CreERT2 (Semerci et al., 2019), and a ROSA tdTomato (TDT) Cre reporter
(Madisen et al., 2010) to lineage trace supporting cells, as well as Atoh1-GFP (Rose et
al., 2009) to label hair cells and supporting cells undergoing transdifferentiation into new
52
hair cells (Tao et al., 2021). Lineage-traced TDT+ supporting cells that transdifferentiate
into hair cells upon DAPT treatment up-regulate hair cell-specific transcription factors
Atoh1-GFP and POU4F3 (Figure 4.1A and Figure 4.2A). Thus, supporting cells
undergoing transdifferentiation exhibit TDT+/ GFP+ double labeling, whereas supporting
cells not responding to DAPT are only labeled with the TDT reporter (Figure 4.1A, B).
Cochlear sensory epithelium was explanted and cultured with or without DAPT for 48
hours, followed by enzymatic digestion and FACS purification for single-labeled
nonresponsive TDT+ supporting cells and double-labeled TDT+/GFP+
transdifferentiated supporting cells (Figure 4.1C). We processed these purified cells for
bisulfite treatment for WGBS analysis. We also performed this experiment in supporting
cells at P6 when the supporting cells are no longer responsive to DAPT.
To establish a baseline, we compared the CpG methylation percentage at de
novo hair cell-specific DMRs to endogenous P1 supporting cells (Figure 4.1D, left).
Upon DAPT treatment, only successfully transdifferentiated supporting cells at P0
showed a decrease in mCpG percentage at de novo hair cell-specific DMRs (Figure
4.1D, middle). No decrease in mCpG percentage was observed at the pre-established,
de novo supporting cell-specific, or de novo common DMRs, suggesting that the
demethylation is highly specific to the hair cell-specific DMRs (Figure 4.2B).
Nonresponsive TDT+ supporting cells in both the P0 and P6 cochlear explants had
comparable percentage of mCpG to baseline in vivo P1 supporting cells. Quantifying
the change, we found a 10% decrease in mCpG percentage from 72.367% in
53
nonresponsive TDT+ supporting cells to 62.333% in transdifferentiated TDT+/GFP+
supporting cells (p-value: 2.966e-61; Figure 4.1E, top). The 10% decrease in DNA

54

55
Figure 4.1. DAPT-mediated transdifferentiation of SCs induces de novo
hypomethylation of HC-specific enhancers.
(A) P0 and P8 cochlear explant cultures were treated with either DMSO or 10µM DAPT
to induce supporting cell transdifferentiation. Simultaneously, cochlear explants were
treated with 0.02 mg/ml of (Z)-4-Hydroxytamoxifen to induce Lfng-CreER-mediated
tdTomato (TDT) labeling of supporting cells from the ROSA-Ai9 Cre reporter mouse.
Atoh1-GFP reporter mice were used to label both original and transdifferentiated hair
cells. Transdifferentiated hair cells were identified with double labeling of TDT and GFP.
Cultures were induced for 2 days. Scale bar: 100µm. (B) Cross section of P0 cochlear
explant treated with either DMSO or 10 µM DAPT. TDT-labaled supporting cells that
transdifferentiated into GFP+ hair cells are labeled yellow. (C) FACS gating strategy for
purifying TDT+ (red) only and TDT/GFP+ (yellow) supporting cells. The x-axis
represents GFP fluorescence; the y-axis represents tdTomato fluorescence. (D)
Average of mCpG percentage over HC-specific DMRs after DAPT treatment at P0 and
P6. P1 original HC and SC were used to compare to baseline. P0 cochlear explants
contained both TDT+ only and TDT/GFP+ supporting cells, whereas P6 cochlear
explants only contained TDT+ supporting cells after DAPT treatment. (E) Average
mCpG percentage over de novo HC-specific DMRs and de novo common DMRs were
summarized in a box plot to calculate statistical significance using the Wilcoxon Rank
Sum test (p-value: 2.966e-61). DMRs were extended out to ±500 bp windows. (F) P0
cochlea explant cultures were simultaneously treated with 10 µM DAPT and 0.02 mg/ml
of (Z)-4-Hydroxytamoxifen as in (A). Additionally, cochlear explants were treated with
the TET inhibitor Bobcat339 at 90µM, 180 µM, and 270 µM. After 2 days of culture,
cochlea explants separated into apex and base regions, dissociated, and quantified by
FACS as in (B). Percentage permissive supporting cells were calculated as the number
of TDT/GFP+ supporting cells over total TDT+ supporting cells. n=4 replicates per
condition. p-value < 0.05: *; p-value < 0.01: **.

56
methylation is notable because the demethylation process occurred within a relatively
brief window of 48 hours after DAPT exposure. In addition, earlier studies demonstrate
that 5-10% DNA methylation differences can be biologically significant, for example in
the case of the Oct4 locus between human embryonic stem cells, their derived cell
populations, normal somatic tissues, and disease conditions (Hodges et al., 2011; Ziller
et al., 2013). Using de novo common regions as a control, no significant change in DNA
methylation levels was observed (p-value: 1; Figure 4.1E, bottom). Since the
transdifferentiating TDT+/GFP+ supporting cells are postmitotic, cell-cycle dependent
demethylation through the loss of DNMT1 maintenance methylase is not possible and
demethylation must occur though de novo activity of DNA demethylating TET enzymes
(Monk et al., 1991; Tahiliani et al., 2009; F. Zhang et al., 2007). In summary, activation
of the hair cell GRN in transdifferentiating supporting cells is in part mediated by DNA
demethylation of hair cell-specific DMRs by TET enzyme activity.
To examine whether TET-mediated DNA demethylation is required for supporting
cells to transdifferentiate into hair cells. We treated P0 cochlear explants with 10uM of
DAPT and a dose series of Bobcat339 (Chua et al., 2019), a TET1 and TET2 enzyme
inhibitor, at 90uM, 180uM, and 270uM for 48 hours. Cochleas were then dissociated by
the end of 48 hours for FACS-mediated quantification. We hypothesized that blocking
TET enzyme activity with Bobcat339 would prevent supporting cells from successfully
up-regulating ATOH1 and transdifferentiating in response to DAPT. As a result,
treatment would lead to a decrease in the proportion of double-labeled GFP+/TDT+
cells compared to total TDT+ cells, quantified as percentage of permissive supporting
57
cells. On treating with 10uM DAPT, 55.40% of supporting cells were permissive for
transdifferentiation. The transdifferentiation rate decreased in a dose-dependent
manner with increasing concentrations of Bobcat339 co-treatment (Figure 4.1F).
44.54% permissive supporting cells with 90uM Bobcat339 co-treatment (n = 4, p-value
= 0.032, *), 24.72% permissive supporting cells with 180uM Bobcat339 co-treatment (n
= 4, p-value = 0.026, *), and 13.05% permissive supporting cells with 270uM Bobcat339
co-treatment (n= 4, p-value = 0.004, **). These findings strengthen the conclusion that
supporting cells require TET enzyme activity to demethylate and de-repress hair cell-
specific DMRs to enable activation of target genes during transdifferentiation.

58

Figure 4.2. Effect of DAPT-mediated transdifferentiation on POU4F3 expression and
DNA methylation signal at DMRs.
(A) P0 and P8 cochlear explants treated with either DMSO or 10 µM DAPT for 2 days
were immunostained for the hair cell-specific marker POU4F3. Only P0 cochlear
explants treated with DAPT showed supernumerary POU4F3+ hair cells. (B) Average of
mCpG percentage over pre-established DMRs, de novo SC-specific DMRs, and de
novo common DMRs after DAPT treatment at P0 and P6. There is no evidence of DNA
hypomethylation at these DMRs in supporting cells undergoing transdifferentiation in
response to DAPT.

59
Chapter 5. The epigenetic effects of long-term deafening on adult
mouse cochlear supporting cells
Introduction
DNA methylation is considered to be the final layer of epigenetic silencing to
permanently solidify heterochromatin and make it refractory to transcription factors and
transcriptional machinery (Rountree & Selker, 2010). We have demonstrated that pre-
established CpG island promoters undergo increasing de novo DNA methylation and
decreasing accessibility in postnatal maturing supporting cells (Figure 3D). Further, we
showed that the transdifferentiation of P1 supporting cells both induces de novo DNA
demethylation (Figure 4E) and is attenuated by inhibiting TET enzyme activity (Figure
4F), suggesting that DNA methylation plays a key role in silencing the hair cell gene
regulatory network through heterochromatin formation. Next, we wanted to understand
the implications of this heterochromatinization in the pathological context of hearing
loss. Specifically, we wanted to understand whether there are further changes to the
supporting cell chromatin accessibility landscape in the adult stage, as well as in a long-
term deafened state which shares more features with the clinical context of human
hearing loss. Since supporting cell subtypes are functionally distinct at adult stages, we
used scMultiome to simultaneously profile gene expression and chromatin accessibility
at the single cell level. Comparing accessibility between organ of Corti supporting cell
subtypes allows us to identify cell type-specific enhancers, as well as supporting cells
that are more receptive to conversion into new hair cells.
60
Simultaneous profiling of RNA and ATAC at the single cell level in cochlea
of wildtype and long-deafened mice
We collected scMultiome datasets for cochlear supporting cells in wildtype P1,
P8 (Iyer et al., 2022), and P70 mice, as well as in long-deafened P70 mice (Figure 5A).
Single cell data was clustered using ‘uniform manifold approximation and projection’
(UMAP) and ‘weighted-nearest neighbor’ (WNN) using the Seurat R package, which
integrates both the gene expression and the accessibility information to define a “joint”
cellular state (Hao et al., 2021). WNN analysis is an improvement over previous
methods of clustering that were based on RNA or ATAC data alone by improving our
ability to resolve cell states (SI Appendix, Figure S5A). scMultiomic profiling is
especially well-suited for resolving the diversity of postnatal organ of Corti supporting
cells, which no longer express distinguishing developmental genes and instead share
common morphological and molecular features (Wan et al., 2013).
We annotated the clusters using gene expression data cross-referenced with
previously published literature. At P1, we identified most cell subtypes within the organ
of Corti, such as inner hair cells (iHC), outer hair cells (oHC), inner phalangeal cells
(iPh), pillar cells (PC), Deiters’ cells (DC), Hensen’s cells (Hn), and Claudius cells (Cl).
We also identified cells of the GER, spiral limbus, roof, stria vascularis, and the spiral
ligament. At P8, we similarly identified the following organ of Corti cell subtypes: iHC,
oHC, border cells (BC), iPh, PC, and three distinct populations of Deiters’ cells (DC1,
DC2, DC3), although we could not assign medial-lateral positional identities to these
three populations. Other identified cell types come from the basement membrane (BM),
GER, SV, Spiral ganglia neurons (SGN), pericytes (Peri), and endothelium (Endo).
61
Since we FACS-purified Lfng-GFP+ lineage-traced supporting cells for the P70 wildtype
and long-term deafened datasets, we identified a more focused set of organ of Corti cell
subtypes labeled by this reporter: BC, iPh/IP, DC1, DC2, DC3 (see methods). The P70
UMAP projection displays the integrated P70 wildtype and P70 long-term deafened
datasets (SI Appendix, Figure S5B).
We separated chromatin accessibility (ATAC) signal by cell types, and quantified
it over the pre-established, hair cell-specific, supporting cell-specific, and common
DMRs (Figure 5.1B). Collectively, supporting cell subtypes showed progressively
decreasing accessibility at pre-established and hair cell-specific DMRs between P1, P8,
and P70 time points. Interestingly, accessibility increased in the P70 long-term
deafened supporting cells compared to the P70 wildtype supporting cells, specifically at
hair cell-specific DMRs in BC, PC, and DC2 clusters (Figure 5.1B). An example of this
can be seen at the Myo7a gene locus (Figure 5.1C). This suggests that the loss of hair
cells may cause supporting cells to modify their chromatin structure following damage,
which may allow for easier re-activation of the hair cell GRN in the deafened state.
Although we did not see a collective increase in accessibility over the 3,058 SC-
specific DMRs, we did see an increase at some specific regions. For instance, the Gjb2
and Gjb6 loci show increased accessibility in P70 supporting cell subtypes, which are
genes that encode gap junction proteins responsible for shuttling potassium ions and
maintaining endocochlear potential (Figure 5.1D, right). Coupled with the gradual loss of
accessibility at the Atoh1 locus (Figure 5.1D, left), we see further evidence for gene
program switching in the postnatally maturing supporting cells, where the hair cell gene
62
program is silenced in favor of a differentiated and functional supporting cell gene
program. Additional examples of hair cell-specific genes Pou4f3, Jag2, Dll1, and Myo7a,
and supporting cell-specific genes Sox2, Notch1, and Jag1 are shown in Figure 5.2A
and Figure 5.3B, respectively.
We also find that chromatin accessibility shifts to a more accessibility state in the
long-term deafened supporting cells compared to wildtype supporting cells at hair cell-
specific enhancers. Corroborating our ATAC boxplot data, we find that the border cell
supporting cell subtype demonstrates the most dramatic shift in ATAC accessibility, with
a correlation coefficient R of 0.52 (p < 2.2e
-16
) (Figure 5.4A). To connect accessibility to
gene expression, we performed linkage analysis between our single nuclei gene
expression dataset and our single nuclei ATAC dataset and found strong linkage at the
Myo7a gene locus (Figure 5.5B). Specifically, we find increased accessibility in the
Border cell supporting cell subtype once again, which correlates with increased
detection of Myo7a mRNA transcripts (Figure 5.5A). We do not find increased in either
chromatin accessibility at the Myo7a locus or Myo7a mRNA in any other supporting cell
subtypes. This suggests that the border cell may be the ideal candidate for
reprogramming efforts, especially in the long-deafened context, where we observe
some modest relaxing of the chromatin landscape at some hair cell genes.

63

64
Figure 5.1. Single cell multiomic profiling of mouse cochlear cell types during
developmental, adult homeostatic, and deafened states.
(A) Weighted nearest neighbor (WNN) clustering and UMAP of P1, P8, P70 wildtype,
and P70 long-deafened cochlear cells. P1 and P8 data were obtained from Iyer et al.,
(2022). Distinct clusters of cochlear cell subtypes can be identified at each age. oHC:
outer hair cells; iHC: inner hair cells; DC: Deiters’ cells; OF/SL: otic fibrocytes/spiral
ligament cells; iPH: inner phalangeal cells; Hn: Hensens’ cells; PC: pillar cells; Cl:
Claudius cells; SP: spiral prominence; Roof: cells of the cochlear roof; MGER: medial
greater epithelial ridge; LGER: lateral greater epithelial ridge; SL: spiral limbus; BC:
border cells; IP: inner pillar cells; SV: stria vascularis; SGN: spiral ganglion neurons;
Endo: endothelial cells; Per: pericytes; BM: basement membrane. (B) Box plots of
average ATAC normalized reads from each organ of Corti cell subtype over DMRs (****,
p-value < 0.0001, Wilcoxon Rank Sum test). (C) Representative genome track of ATAC
signal at the Myo7a locus. Supporting cell subtypes were chosen based on observable
change between wildtype and long-deaf conditions in (B). HC-specific DMR regions are
shown for reference. Highlighted regions show gain of ATAC peaks in deafened
supporting cells compared to wildtype. (D) Representative genome track of ATAC signal
from organ of Corti cell subtypes at the hair cell-specific Atoh1 and supporting cell-
specific Gjb2 and Gjb6 loci. Highlighted regions show loss or gain of ATAC peaks as
supporting cells mature between P1 and P70.

65

Figure 5.2. Integrating multimodal single cell datasets between RNA and ATAC, and
between P70 wildtype and long-deafened conditions.
(A) UMAP projections of P1, P8, P70 wildtype, and P70 long-deafened cochlear cells
based on RNA gene expression and ATAC chromatin accessibility. (B) Contribution of
P70 wildtype and P70 long-deafened cells to clusters after integration of single cell
multiome datasets.
66

Figure 5.3. Single cell ATAC-seq profiling of key hair cell and supporting cell genes.
(A) Examples of ATAC peaks of each organ of Corti cell subtype at the loci of key hair
cell genes Pou4f3, Jag2, Dll1, and Myo7a. (B) Examples of ATAC peaks of each organ
of Corti cell subtype at the loci of key supporting cell genes Sox2, Notch1, and Jag1.

67

Figure 5.4. ATAC signal shifts to a more accessibility state in long-term deafened
supporting cells compared to wildtype at hair cell-specific enhancers.
(A) Log2 transformation of RPGC normalized ATAC reads over hair cell-specific
enhancers shows a population increase in accessibility in the deafened state compared
to wildtype state. R = 0.52, p < 2.2e
-16
.

68

Figure 5.5. Linkage between accessibility and gene expression of Myo7a gene show
increased expression in deafened state compared to wildtype.
(A) Increased accessibility at the hair cell-specific Myo7a gene correlates with increased
in Myo7a mRNA expression in Border cells (BC) in the deafened state compared to
wildtype. (B) Linkage analysis reveals potential Myo7a enhancers with correlation
between accessibility and gene expression.

69
Conclusion
As cells transition from totipotent and pluripotent states to different cell lineages
in development, gene networks are activated to convert cells to progressively more
restricted cell identities. Concurrently, repressive epigenetic barriers are deployed to
silence gene networks of alternative cell lineages and cell fates. This maturation
process is critical for maintaining the fidelity of tissue function and preventing the
uncontrolled growth of cancers. The processes of terminal differentiation and maturation
frequently militate against regeneration in mammals, and this age-dependent decline in
regenerative potential has been well characterized in tissues such as skin, brain, heart,
and muscle (Day et al., 2010; Gurtner et al., 2008; B. Liu et al., 2022; López-Otín et al.,
2013; Porrello et al., 2011).
Like cells in many other tissues, cochlear mechanosensory hair cells do not
regenerate in adulthood, and demonstrate only a limited regeneration potential in the
first postnatal week of mice through direct transdifferentiation of neighboring supporting
cells (Korrapati et al., 2013; White et al., 2006; Yamamoto et al., 2006; Zhao et al.,
2011). Previous studies have characterized transcriptional and epigenetic changes that
recapitulate this transition from regenerative ability to functional maturation and loss of
plasticity (Maass et al., 2016; Stojanova et al., 2015; Tao et al., 2021). One mechanism
to silence hair cell gene regulatory networks in supporting cells is through enhancer
decommissioning, where enhancers lose H3K4me1 priming modifications (Tao et al.,
2021). H3K4me1 enhancer priming is responsible for the recruitment of nucleosome
remodelers or pioneer transcription factors (Bogdanović et al., 2012; Zaret & Carroll,
70
2011), the loss of which may explain why the hair cell gene networks cannot be
activated in mature supporting cells.
Despite the importance of enhancer decommissioning, it is notable that
decommissioned enhancers are not permanently silenced: they can still be primed and
reactivated if the CpG dinucleotides within the enhancer region are retained in a
hypomethylated state (Jadhav et al., 2019; Kim et al., 2010; Polo et al., 2010). A
combination of enhancer decommissioning, acquisition of repressive histone marks and
DNA methylation is therefore more likely to place lineage-specific genes in a more
inaccessible state. In the present study, we demonstrated that some enhancers bound
by the hair cell transcription factor ATOH1 that reside in DNA methylation “valleys” of
hair cell-specific genes are hypermethylated as supporting cells undergo maturation
(Figure 2C). In regenerative tissues such as the intestines, these developmental
enhancers maintain a DNA demethylated state to allow for fast redeployment of
developmental genes within the Lgr5+ stem cell population (Jadhav et al., 2019). In
contrast, the hypermethylation of hair cell CpG island promoter genes may explain why
the cochlea demonstrates poor regenerative abilities compared to the intestines, which
is hailed as being one of the most regenerative tissues (Hageman et al., 2020; Y. Liu &
Chen, 2020). Other examples of DNA accumulating during postnatal maturation
involves the hypermethylation and subsequent silencing of tumor suppressor genes in
many cancers (Esteller, 2002, 2007). This work documents the first example of DNA
hypermethylation as a mechanism to restrict cell states during late stage differentiation.
71
DNA methylation of CpGs is a mechanism to permanently silence regulatory
elements by either creating steric hindrance between the transcription factor and its
binding motif (Domcke et al., 2015; Yin et al., 2017) or by promoting chromatin
condensation and heterochromatin formation (Buitrago et al., 2021; Rountree & Selker,
2010). Moreover, we show evidence of heterochromatin formation, where hair cell
enhancers that have acquired H3K27me3 repressive marks further transition to CpG
methylation-mediated silencing, accompanied by complete loss of chromatin
accessibility (Figure 3E-H). Thus, for a subset of enhancers, hypermethylation of hair-
cell specific genes is sufficient for permanent silencing in mature supporting cells. We
postulate that this heterochromatin formation impedes ATOH1 binding and prevents, at
least in part, the activation of the hair cell gene program in mature supporting cells.
Ultimately, DNA methylation silences genes of alternative cell fates and restricts
pluripotency in progenitor cells as they differentiate and mature (Domcke et al., 2015;
Meissner et al., 2008; Mohn et al., 2008). In the case of maturing supporting cells, this
can be seen as a transition from a plastic, progenitor-like state to a state where
transdifferentiation potential is lost (Bramhall et al., 2014; Shi et al., 2012).
In addition to supporting cells DNA methylating hair cell loci with age, we also
observed evidence for active regulation of DNA methylation of some hair cell loci in
young supporting cells. Of note, we found that neonatal supporting cells undergoing
transdifferentiation in the presence of the Notch inhibitor DAPT show de novo DNA
demethylation at a set of activated hair cell-specific enhancers (Figure 4C) (Tao et al.,
2021), suggesting that DNA methylation is already present at some hair cell loci in
72
young supporting cells, but that this methylation is reversible. TET enzymes promote
active DNA demethylation through a cell-cycle independent mechanism (J. U. Guo et
al., 2011; Tahiliani et al., 2009; T. Wang et al., 2012), and are necessary for proper
differentiation during embryonic development (Dawlaty et al., 2014; Lan et al., 2021; L.
Wang et al., 2022), making them an excellent candidate for removing DNA methylation
in postmitotic supporting cells. Significantly, we show that inhibiting TET enzyme activity
is sufficient to prevent transdifferentiation of young supporting cells (Figure 4C).
Whether Atoh1 is actively recruiting TET enzymes to hair cell-specific enhancers in
supporting cells is unclear and follow up experiments would be needed to determine
this. Regardless, these data show that DNA methylation is already being deployed to
silence hair cell enhancers in young supporting cells, but that it is plastic, such that DNA
demethylation of these enhancers is required for transdifferentiation into hair cells.
Evidence of age-related epigenetic changes manifests in a multitude of ways,
such as by the loss of regenerative capacity in normally highly regenerative tissues
(Day et al., 2010; Gurtner et al., 2008; B. Liu et al., 2022; López-Otín et al., 2013;
Porrello et al., 2011) or by the formation of cancers (Baylin & Jones, 2016). In addition
to characterizing changes in DNA methylation as supporting cells mature, we also
sought to understand how the chromatin epigenetic landscape continues to change in
the adult, well beyond this developmental period. Our scRNA-seq and scATAC-seq
multiomic analysis shows that all cochlear supporting cell types continue to lose
chromatin accessibility at hair cell-specific enhancers near CpG island promoter genes
over the first 10 weeks of postnatal life (Figure 5B). Interestingly, we showed that mice
73
deafened for six weeks showed partial recovery of chromatin accessibility at these
enhancers, an example of which is the Myo7a locus (Figure 5C), suggesting that they
may be potential candidates to drive expression of hair cell-specific genes by
regenerative therapies. One possible direction for future studies in re-establishing the
hair cell gene regulatory network might involve targeting chromatin remodelers to these
hair cell enhancers. Other groups have demonstrated that the activation of the SWI/SNF
chromatin remodeling enzyme SMARCA4/BRG1 is required to initiate oligodendrocyte
differentiation in mature animals (Y. Yu et al., 2013). One possible application of this in
human hearing loss could be the activation supporting cell enhancers at the Gjb2 and
Gjb6 loci (Figure 5D) in adults as an alternative to AAV gene therapy strategies for
DFNB1 hearing loss (Crispino et al., 2011; J. Guo et al., 2021; D. Wang et al., 2019).
Our current study has shown that DNA methylation of hair cell loci is a correlate
of the failure of adult supporting cells to regenerate hair cells. However, an additional
consideration for hair cell regeneration is whether down-regulation of the mature
supporting cell gene program is required prior to successful regeneration of hair cells
from supporting cells. As cells terminally differentiate, their access to alternative cell
type gene programs become limited (Davie et al., 2018; Wulff & Freshman, 1961; M. J.
Zhang et al., 2021). Forcing the activation of a hair cell gene program – for example, by
transcription factor reprogramming (Iyer et al., 2022; Menendez et al., n.d.) - while the
supporting cell gene program is still active may prove detrimental (Gu et al., 2018;
Jorgensen et al., 2018; Liao et al., 2015). Moreover, although we show some evidence
of hair cell gene loci becoming more accessible in the supporting cells of deafened
74
mice, hair cell loci containing binding sites for key transcription factors responsible for
establishing hair cell identity such as ATOH1 and POU4f3 remain closed in mature
supporting cells (Figure 5D, SI Appendix, Figure S6 A). Thus, directed reprogramming
using hair cell transcription factors need to be accompanied by the manipulation of
epigenetic modifications (J. Chen et al., 2013; Esteban et al., 2010; Marks et al., 2012;
W. J. McLean et al., 2017; T. Wang et al., 2011) for functional regeneration of hair cells
in the deafened cochlea.

75
Materials and Methods

Animals
Experiments were conducted in accordance with the policies of the Institutional Animal
Care and Use Committee of the Keck School of Medicine of the University of Southern
California. Animals for timed matings were put together in the evening, and plugs found
the next morning were denoted as embryonic day 0.5 (E0.5). The first day after birth is
designated as postnatal day 0 (P0) in this study. p27
Kip1
-GFP BAC transgenic reporter
mice were used for E13.5 prosensory progenitor cell collection (Y.-S. Lee et al., 2006).
Atoh1-GFP knock-in mice (MGI:Atoh1
tm4.1Hzo
;C57bl6 background) were used for E17
and P1 hair cell (HC) collection (Rose et al., 2009). Lfng-GFP transgenic mice
(MGI:Tg(Lfg-EGFP)HM340Gsat) were used for P1 supporting cell collection (Gong et
al., 2003). Lfng-CreERT2 (MGI: Tg(Lfng-cre/ERT2)1Mmsa) / Rosa-tdTomato (ROSA-
Ai9, MGI: Gt(ROSA)26Sor
tm9(CAG-tdTomato)Hze
; CD1 background) transgenic mice were
used to lineage trace supporting cells after tamoxifen exposure (Semerci et al., 2017).
Lfng-CreERT2 mice were crossed with NuTRAP mice (MGI: Gt(ROSA)26Sor
tm2(CAG-
NuTRAP)Evdr
) (Roh et al., 2017) for P6, P8, and P21 supporting cell collection. The knock-
in mice are fluorescently labeled at the supporting cell nuclear membrane and nucleolus
with tdTomato and GFP protein, respectively. Lfng-CreERT2 mice were also crossed
with Atoh1-GFP mice to identify double-labeled TDT+/GFP+ supporting cells
undergoing transdifferentiation after DAPT exposure in the cochlear explant culture
experiments. Lfng-CreERT2 NuTRAP mice were crossed with Pou4f3
DTR
(B6.Cg-
76
Pou4f3
tm1.1(HBEGF)Jsto
/RubelJ) mice to lineage trace supporting cells and kill hair cells
after administration of diphtheria toxin.

Epithelial preparation for E13.5 cochleas
For E13.5 cochleas, the inner ear was dissected from the mouse head, followed by
incubation in PBS containing 0.5 U/ml of Dispase II (Sigma D4693) and 275 U/ml of
Collagenase Type 1 (Worthington LS004196) at room temperature for 10 minutes. The
Dispase II and Collagenase were removed and quenched with PBS + 5% Fetal Bovine
Serum (FBS) (Genclone 25-514). The cochlear epithelium was separated from the
mesenchyme and dissociated into single cells.

Sensory epithelium dissection for P1, P6, P8, P21, and P70 cochleas
Cochlear microdissections were performed using a stereo microscope. The inner ear
was extracted and placed in a dish containing ice-cold modified artificial cerebrospinal
fluid solution (ACSF) [119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1 mM
NaH2PO4, 26.2 mM NaHCO3, 10mM HEPES, 11mM D-Glucose] on top of an ice block.
Cartilage and bone were removed to expose the cochlear epithelium. Finally, the
cochlear epithelium was separated from the spiral ganglion and modiolus, and then
placed into a separate dish containing ACSF on ice until all ears were processed.

77
Cochlear explant culture and drug treatment
For explant culture, the cochlear sensory epithelium was further dissected to remove
the lateral wall and cultured on SPI black filter membranes (Spi supplies). DMEM:F12
media (Gibco 11330-032) was used to culture P0 cochlear explants, whereas L15
Leibovitz media (Gibco 11415-064) buffered with 10mM HEPES was used to culture P6
and older cochlear explants. Explant culture medium was supplemented with 1x N2
(Gibco 17502-048), 1x B27 (Gibco 17504-044), 100 U/ml Penicillin (Sigma P3032), 1x
GlutaMAX (Gibco 35050061), 1mM N-acetyl cysteine, 10ng/ml Stembeads EGF
(Stemcultures SBEGF), 10ng/ml Stembeads FGF (Stemcultures SB500), which was
made fresh weekly. To initiate Lfng-CreER-mediated lineage tracing of supporting cells,
a final concentration of 0.02 mg/ml of (Z)-4-Hydroxytamoxifen (Sigma H7904) was
added to the culture media. For transdifferentiation experiments, DAPT (Calbiochem
565770) was added at 10 µM to the culture media. In the TET inhibition experiments,
Bobcat339 (Sigma SML2611) was added at between 90-270µM to the culture media. In
every explant culture experiment, (Z)-4-Hydroxytamoxifen, DAPT and Bobcat339 were
added concurrently at the start of the culture.

Single cell dissociation for E13.5, P1, P8, and P21 cochleas
After cochlear sensory epithelia were isolated, they were incubated in 500 µl of
0.25% Trypsin-EDTA (Gibco 25200-056) at 37°C for 15 minutes. For P21 enzymatic
dissociation to compare with the NuTRAP nuclei isolation method, cochlear sensory
epithelia were incubated in a cocktail of 400μl of 0.25% Trypsin-EDTA (Gibco 25200-
78
056), 50μl of 5U/ml Dispase II (Sigma D4693), and 50μl of 2750 U/ml Collagenase I
(Worthington-Biochem LS004196) for 15 minutes at 37 °C. The tissue was dissociated
into a single-cell suspension by trituration with a P200 pipette for 2 minutes. Trypsin
was quenched with 50µl of FBS. The single-cell suspension was filtered using a Falcon
5ml round bottom polystyrene test tube with a 40µm cell strainer snap cap (Corning
352235), followed by centrifugation at 500 x g and 4°C for 5 min. The supernatant was
decanted, and the pellet was resuspended in PBS with 5% FBS for FACS purification.

Nuclei isolation for P6, P8, P21, and P70 cochleas
Cochlear sensory epithelium was isolated from the spiral ganglion with the stria
vascularis kept intact due to the supporting cells being easily separated along with the
stria. The sensory epithelium and stria were placed into a 1ml Dounce tissue grinder
(Wheaton 357538) on ice. Carry-over ACSF was removed. 1 ml of chilled
homogenization buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 20 mM HEPES-KOH
pH 7.9, 20% glycerol). Before use, the following components were added at their
respective concentrations: 1mM DTT (Thermo Scientific R0861), 0.5mM Spermidine
(Sigma S0266), 1% BSA (Sigma A4503), and 1x cOmplete EDTA-free protease inhibitor
cocktail (Roche 11836170001). The cOmplete EDTA-free protease inhibitor comes as a
tablet, which was dissolved in 1ml of dH2O to make a 50x solution that was stored at
4°C for 1 week. NP-40 (Calbiochem 492015) was spiked into the Dounce tissue grinder
at a final concentration of 0.3%. The tissue was homogenized with a chilled loose pestle
by first pressing the pestle down and turning to grind the tissue, followed by quick
79
upstrokes 10 to 15 times to create cavitation. It was critical to not lift the Dounce pestle
above the liquid layer to reduce bubbles. This process was repeated with a chilled tight
pestle. Although some epithelial tissue was sometimes still visible, no further
homogenization was performed as this may jeopardize the integrity of the already-
released nuclei. The homogenate was filtered using a Falcon 5ml round bottom
polystyrene test tube with a 40 µm cell strainer snap cap and spun down at 1000 x g
and 4°C for 10 minutes. The supernatant was decanted, and the pellet was
resuspended in 1ml of nuclei wash buffer (NWB) (20 mM HEPES-KOH pH 7.5, 150 mM
NaCl, 0.1% NP-40). Before use, the following components were added at the respective
concentrations: 1mM DTT, 0.5mM Spermidine, 2% BSA, and 1x cOmplete EDTA-free
protease inhibitor cocktail.

FACS purification
Fluorescence-activated cell sorting (FACS) of cells or nuclei was performed on
either an Aria I, Aria II, or FACSymphony (BD Biosciences). For CUT&RUN and
CUT&Tag, cells or nuclei were sorted into NWB. For WGBS, cells or nuclei were sorted
into lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.01% Triton X-
100). For Single Cell Multiome ATAC + Gene expression (10x Genomics), cells or
nuclei were sorted into PBS and 0.1% BSA.

80
Cryosectioning, Immunostaining, and microscopy
Cultured cochlear explants were fixed on the SPI membrane with 4%
paraformaldehyde (Electron Microscopy Sciences) at room temperature for 5 min. For
cryosections, fixed organs were cryoprotected in 30% sucrose until sunk, embedded in
Tissue-Tek OCT (Sakura 4583), and frozen on dry ice. Organs were sectioned in a
Leica cryostation CM3050S at 10µm thickness. Sections or whole mount preparations
were simultaneously permeabilized and blocked with a blocking buffer (1% Triton X-
100; 5% normal donkey serum in PBS) at 4°C overnight. On the second day, cochlear
explants were incubated with chicken anti-GFP primary antibody (Genetex GTX13970)
at 1:1000 dilution and DAPI (Biolegend 422801) at 1:10,000 dilution in a blocking buffer.
On the third day, cochlear explants were washed three times with PBS for 10 minutes
on a shaker at low speeds. Afterwards, cochlear explants were incubated with a
secondary antibody diluted at 1:1000 in blocking buffer for 2 hours at room temperature.
Finally, cochlear explants were washed three times with PBS before mounting onto
glass slides. Images were taken with a Zeiss LSM780 confocal microscope. The
following endogenous signals did not need to be amplified with antibody staining: Lfng-
CreERT2 NuTRAP, Lfng-CreERT2 Rosa-tdTomato.

Whole genome bisulfite sequencing (WGBS) library preparation
CpG methylation was detected using bisulfite conversion followed by WGBS. At
least 8,000 cells or nuclei were used as input per replicate, and 2 or 3 biological
replicates were prepared for each stage or condition. Briefly, cells or nuclei were FACS
81
purified into lysis buffer (10mM Tris pH 7.5, 100mM NaCl, 1mM EDTA, and 0.01%
Triton X-100). Proteinase K (Qiagen 19133) was added to a final concentration of 3
mAU/ml into the lysate and digested at 65°C for at least 1 hour. gDNA was purified
using the Genomic DNA Clean & Concentrator-10 kit (Zymo Research D4010). gDNA
concentration was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen Q32854).
Unmethylated Lambda DNA (Promega D1521) was spiked in at 0.5% of input gDNA
amount. Libraries were prepared using the Pico Methyl-Seq Library Prep Kit (Zymo
Research D5456). Libraries were pooled and sequenced for 150 cycles in paired-end
mode on the Novaseq 6000.
CUT&RUN
Genome-wide transcription factor binding was detected using CUT&RUN
according to the original protocol (Skene & Henikoff, 2017) following the high Ca
2+
/low
salt modification. At least 10,000 cells or nuclei were used as input per replicate, and at
least two biological replicates were prepared for each stage or condition. The following
antibodies were used: rabbit anti-GFP (Torrey Pines Biolabs, TP401) to detect ATOH1
binding, mouse anti-POU4F3 (Santa Cruz, sc-81980) to detect POU4F3binding. Rabbit
anti-mouse IgG (Abcam ab46540) was used as the primary antibody for negative
controls. Rabbit anti-mouse antibody (Abcam, ab46540) was used as the secondary
antibody. DNA fragments extracted from CUT&RUN samples were made into
sequencing libraries using the xGen DNA Library Prep Kit with UMI Adapters (Idt
10005903) with 15 cycles of amplification. Library concentration and fragment
distribution were measured on the Agilent Tapestation using a High Sensitivity D5000
82
ScreenTape (Agilent 5067-5592). Libraries were size-selected for DNA fragments
greater than 150 bp using SPRISelect beads (Beckman Coulter B23318). Libraries were
pooled and sequenced for 37 cycles in paired-end mode on the Illumina NextSeq 500.

CUT&TAG
Histone modifications were detected using CUT&Tag according to the original
protocol (Kaya-Okur et al., 2019). Briefly, at least 2,000 cells or nuclei were used as
input per replicate, and at least two biological replicates were prepared for each stage
or condition. The following antibodies were used: Rabbit anti-H3K4me1 antibody (Active
Motif 61633), rabbit anti-H3K4me2 antibody (Epicypher 13-0027), rabbit anti-H3K4me3
antibody (Active Motif 39159), rabbit anti-H3K27me3 antibody (Active Motif 39155). The
guinea pig anti-Rabbit IgG antibody (Antibodies-online.com ABIN101961) was used as
the secondary antibody. Tn5-tagmented DNA fragments were amplified using NEBNext
HiFi 2x PCR Master Mix, a universal i5 primer and a uniquely barcoded i7 primer for 14
cycles. Library concentration and fragment distribution were measured on the Agilent
Tapestation using a High Sensitivity D5000 ScreenTape (Agilent 5067-5592). Libraries
were size-selected for DNA fragments greater than 150 bp using SPRISelect beads
(Beckman Coulter B23318). Libraries were pooled and sequenced for 37 cycles in
paired-end mode on the Illumina NextSeq 500.

83
P70 wildtype and long-term deafened mouse model for scMultiome
We used a Lfng-CreERT2;NuTRAP;Pou4f3
DTR
mouse to lineage trace SCs and
specifically kill hair cells after diphtheria toxin treatment. The P70 long-deafened mice
received 100 mg/kg tamoxifen (Sigma-Aldrich T5648) at P21, 0.01 mg/kg Diptheria toxin
(Sigma-Aldrich D0564) at P28, and allowed to mature to P70, at which point cochlear
tissues were harvested for input into a scMultiome reaction.

scMultiomic sample processing
The Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit was
used to prepare libraries for simultaneously profiling of RNA and ATAC at the single cell
level (10x Genomics 1000285). For P1 and P8 scMultiomic datasets, wild type mice in a
mixed background of CD1 and FVB/NJ, and C57BL/6 were used. Unsorted single cell
suspensions were prepared as described above. For P70 wild type and P70 long-
deafened cochleas, nuclei were isolated as described above NuTRAP-labeled
supporting cell nuclei were FACS purified and used as input. Both single cell and single
nuclei suspensions were centrifuged at 500 x g for 5 min at 4°C, and then processed
according to the 10 x Genomics protocol ‘Nuclei Isolation from Complex Tissues for
Single Cell Multiome ATAC + Gene Expression Sequencing’ (CG000375, Rev B).
Briefly, the supernatant was decanted, and the cell pellet was resuspended in 1ml of
NP-40 Lysis Buffer and incubated for 5 minutes on ice. Nuclei were filtered using a
Falcon 5ml round bottom polystyrene test tube with a 40µm cell strainer snap cap, and
centrifuged at 500 x g for 5 minutes at 4°C. The supernatant was decanted, and nuclei
84
were resuspended in 1ml of PBS + 2% BSA and incubated for 5 minutes at 4°C. Nuclei
were centrifuged at 500 x g for 5 min at 4 °C, resuspended in 100μl of 0.1 x Lysis
Buffer. The pellet was resuspended by pipetting up and down. Nuclei were incubated for
2 minutes on ice and 1ml Wash Buffer was added. The suspension was pipet-mixed. A
sample of the suspension was taken at this point to quantify cell concentration. The
remaining nuclei suspension was centrifuged at 500 x g for 5 minutes at 4°C. The nuclei
pellet was resuspended in the appropriate volume of Diluted Nuclei Buffer to achieve
approximately 2000 nuclei/µl for input into the Single Cell Multiome ATAC +Gene
Expression protocol (10x Genomics, Rev E). For the P1 cochlea, 6065 quality nuclei
data points were recovered. For the P8 cochlea, 9645 quality nuclei data points were
recovered. For the P70 wildtype cochlea, 396 quality nuclei data points were recovered.
For the P70 long-term deafened cochlea, 754 quality nuclei data points were recovered.

scMultiomic data processing
Raw sequencing data from both RNA and ATAC P1 and P8 libraries in fastq
format were used as input into cellranger-arc count (10x Genomics, v2.0.0) for
simultaneous alignment against the mouse mm10 genome. The cellranger-arc output
files ‘filtered_feature_bc_matrix.h5’ and ‘atac_fragments.tsv.gz’ were used as input into
Seurat v4.1.0 for standard quality control pre-processing and filtering out poor-quality
nuclei. This resulted in 4,882 nuclei for P1, 7,049 nuclei for P8, 316 nuclei for P70
wildtype, and 554 nuclei for P70 long-deafened. scMultiome datasets were clustered
using ‘uniform manifold approximation and projection’ (UMAP) and ‘weighted-nearest
85
neighbor’ (WNN) from Seurat, which integrates both the gene expression and the
accessibility information to define a “joint” cellular state (Hao et al., 2021). Datasets
were also clustered based on RNA and ATAC for comparison (SI Appendix, Figure S6
A). Clusters were assigned cell type identities based on known cell markers. For the P8
dataset, clusters identified as spiral ganglia neurons, roof cells, and spiral prominence
cells were removed because they were driving the majority of the differences between
the clusters. Afterwards, P8 cells were re-clustered and re-assigned cell type identity.

Generation of “pseudobulk ATAC” signal from scMultiome
ATAC-seq “pseudobulk” datasets were obtained from scMultiome datasets by
extracting ATAC reads from clusters pertaining to the organ of Corti cell types, and
merging the reads between all cells within the cluster. Briefly, a list of cell barcodes
were obtained for each cluster. Reads matching the cell barcodes were extracted from
aligned BAM files. Representative signal tracks were visualized in IGV v2.4.14.
Pseudobulk ATAC reads per genomic content (RPGC) over DMR sets were visualized
using boxplots. ATAC signal was merged between supporting cell subtypes of the same
stage (for instance, merge all P1 supporting cell subtypes) to determine statistical
significance using the Wilcoxon Rank Sum Test due to the data following a non-
parametric distribution as determined by the Shapiro-Wilk Normality test.

86
NGS Alignment and Data Analysis
For ATOH1 CUT&RUN, libraries were sequenced at 37 base pairs paired-end.
Reads were trimmed based on bp quality and aligned to the GRCm38/mm10 genome
assembly using the STAR aligner (Dobin et al., 2013). UMI-tools (Smith et al., 2017)
was used to remove duplicate reads based on the unique molecular identifiers (UMIs)
that were incorporated during library construction. Peaks were called using MACS2
(Feng et al., 2012) with FDR < 0.01 and --no lambda for individual replicates. Bedtools
(Quinlan & Hall, 2010) was used to filter for common peaks with at least 10% reciprocal
overlapping regions between replicates; common peaks were used to re-center DMRs.
BigWig files were generated using the deepTools bamCoverage command (Ramírez et
al., 2014, 2016) with 50 bp bin size, 150 bp smoothing window, and RPGC
normalization.
For histone modification CUT&Tag, libraries were sequenced at 37 base pairs
paired-end. We processed CUT&Tag data using the CUT&Tag Data Processing and
Analysis pipeline from the Henikoff lab (Zheng et al., 2020). Briefly, reads were hard-
trimmed to 25bp and aligned to the GRCm38/mm10 genome assembly using bowtie2
(Langmead & Salzberg, 2012) using parameters --local --very-sensitive --no-mixed --no-
discordant --phred33 -I 10 -X 700. Duplicates were removed using Picard (Picard,
Broad Institute, 2014/2022). Next, we applied a normalization factor to each sample
based on the signal over the common peak regions. Briefly, for histones with narrow
peak distributions such as H3K4me1 and CUTAC (H3K4me2), SEACR (Meers et al.,
2019) was used to call peaks with non-normalized IgG control track and stringent
87
threshold, and taking only the top 1% of regions by AUC. For histones with broad peak
distributions such as H3K27me3, MACS2 (Feng et al., 2012; Y. Zhang et al., 2008) was
used to call peaks with the --broad and --q 0.1 options set against an IgG control.
Bedtools intersect was used to filter for only common overlapping peaks between all
samples and replicates. 4,718 shared peaks were called for H3K27me3; 4,869 shared
peaks were called for CUTAC; 4,620 shared peaks were called for H3K4me1.
Alignment count data was quantified over shared peaks using summarizedOverlaps
(GenomicAlignments_1.30.0). calcNormFactors (edgeR v3.36.0) was used to calculate
normalization factors for each sample and scaled to total library size. Samples were
scaled to the normalization factor for visualization in IGV or heatmaps.
For WGBS, libraries were sequenced at 150 base pairs paired-end. Reads were
trimmed with a hard clip of 20bp at both the 5’ and 3’ ends using Trim Galore (Krueger,
2016/2022) and aligned as paired end reads to a bisulfite-converted GRCm38/mm10
genome assembly using Bismark (Krueger & Andrews, 2011) with the options --bowtie2
-N 1 -L 20 --non_directional --unmapped -X 800 --dovetail. Spiked-in lambda DNA was
aligned to a bisulfite-converted J02459.1 lambda genome to assess bisulfite conversion
percentage. Only libraries with >98% bisulfite conversion rate were kept. Unmapped
paired end reads were mapped separately as single end read1 and read2 using
Bismark with the options --bowtie2 --non_directional. Paired end and single end
mapped reads were individually deduplicated using deduplicate_bismark, and then
merged using samtools (htslib v1.7). The methylation status of individual cytosine in the
genome was called using Bismark Methylation Extractor (Krueger & Andrews, 2011).
88
The cytosine methylation and coverage data was used to call differentially methylated
regions (DMR) between samples using methylKit (Akalin et al., 2012) with the following
parameters: only cytosine sites with at least a coverage of 2 were kept, tiling window of
1000 bp, step size of 1000 bp, percent methylation difference of at least 10 (sensitivity),
and a false discovery rate (FDR) of < 0.05. Differential analysis was iteratively called
between two samples from all possible combinations C(4,2) of the following cell types
and stages: E13.5 PG, P1 HC, P1 SC, P21 SC. Bedtools intersect was used to identify
the 4 sets of mutually exclusive DMRs capturing distinct DNA methylation signatures.
DMRs were centered on Atoh1 peaks to be used in downstream analyses.
DNA methylation, CUT&RUN, and CUT&Tag signal were visualized on the
Integrative Genomics Viewer (IGV) (Robinson et al., 2011). Principal Components
Analysis (PCA) was performed using deepTools multiBigwigSummary and plotPCA with
the option --rowCenter set (Ramírez et al., 2014, 2016). Heatmaps and signal profiles
were generated using deepTools computeMatrix and plotHeatmap (Ramírez et al.,
2014, 2016). Mean reads per genomic content (RPGC) over DMR sets were visualized
as boxplots using R, and statistical significance was calculated using the Wilcoxon Rank
Sum test due to the data following a non-parametric distribution as determined by the
Shapiro-Wilk Normality test. Gene ontology analysis was performed using GREAT (C.
Y. McLean et al., 2010). Motif analysis was performed using RSAT peak-motifs
(Thomas-Chollier et al., 2012). Annotatr was used to annotate each DMR to known
genomic features (Cavalcante & Sartor, 2017).
89
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Abstract (if available)

Abstract Mammalian hair cells do not functionally regenerate in adulthood, but can be regenerated at embryonic and neonatal stages in mice by direct transdifferentiation of neighboring supporting cells into new hair cells. Previous work showed loss of transdifferentiation potential in supporting cells is in part due to H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during the first postnatal week. However, inhibiting this decommissioning only partially preserves transdifferentiation potential. Therefore, we explored other repressive epigenetic modifications that may also be responsible for this loss of plasticity. We find supporting cells progressively accumulate DNA methylation at developmentally regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of Atoh1, a key transcription factor for hair cell fate. We further show that DNA hypermethylation replaces H3K27me3-mediated repression of hair cell genes in supporting cells, and is accompanied by progressive loss of chromatin accessibility, suggestive of facultative heterochromatin formation. Significantly, another subset of hair cell loci are hypermethylated in supporting cells, but not in hair cells. TET-mediated demethylation of these hypermethylated sites is necessary for neonatal supporting cells to transdifferentiate into hair cells. Finally, we observe changes in chromatin accessibility of supporting cell subtypes at the single cell level with increasing age. Specifically, gene programs promoting sensory epithelium development loses chromatin accessibility, in favor of gene programs that promote physiological maturation and function of the cochlea. Surprisingly, we find chromatin accessibility is partially recovered in a chronically deafened mouse model, which holds promise for future translational efforts in hearing restoration.

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Creator Nguyen, John Duc (author)

Core Title DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Development, Stem Cells and Regenerative Medicine

Degree Conferral Date 2023-05

Publication Date 04/27/2023

Defense Date 04/21/2023

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

Tag cochlea,DNA methylation,hair cells,OAI-PMH Harvest,Regeneration,supporting cells

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Crump, Gage Dekoeyer (committee chair), Gnedeva, Ksenia (committee member), Groves, Andrew Kelton (committee member), McMahon, Andrew P (committee member)

Creator Email jdnukem@gmail.com,johndngu@usc.edu

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Legacy Identifier etd-NguyenJohn-11734

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