University of Southern California Dissertations and Theses

Elucidating the functional role of CHD7 associated nuclear PDH complex and other associated proteins on neural crest development

(USC Thesis Other)

Elucidating the functional role of CHD7 associated nuclear PDH complex and other associated proteins on neural crest development

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content 1

Elucidating the functional role of CHD7
associated nuclear PDH complex and other
associated proteins on neural crest
development

By
Uma Meenakshi Sundaram
1

Mentor: Ruchi Bajpai
1,2

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2016

1 Department of Biochemistry & Molecular Biology, Keck School of Medicine, University of
Southern California, 90033 CA, USA
2: Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, USC

2
ACKNOWLEDGMENTS
I would like to express my gratitude to my advisor, Dr. Ruchi Bajpai, who constantly encouraged
and guided me in researching and writing this thesis. I would like to thank her for giving me this
opportunity to participate in her nascent and vibrant research. I would also like to thank my other
thesis committee members, Dr. Pragna Patel and Dr. Jian Xu, for their guiding comments and
suggestions.

I would like to thank all my lab members Susan Smith, Kaivalya Shevade, Jennifer Oki, Annie
Lynch, Yuhan Sun, Erin Moran, Casey Griffin, Candida Toribio and George Tseng for being a
helpful and supportive team.

I would also like to express my special thanks to the USC Stem cell core for helping me carry
out the FACS sort analysis efficiently.

Thank you everyone for your help and support!

3
TABLE OF CONTENTS:

ACKNOWLEDGMENTS 2
TABLE OF CONTENTS 3
ABSTRACT 5
INTRODUCTION 6
CHD7 and CHARGE syndrome 7
Pyruvate dehydrogenase complex 11
The Human Stem cell differentiation model 14
MATERIALS AND METHODS 16
Tissue culture growth conditions 16
Embryonic stem cell lines 16
Imaging 19
FACS sorting 19
Immunoprecipitation and Western Blotting 19
RESULTS 20
I. Assessing role of PDH and associated protein complexes on NCC
formation using inducible knockdown technology
20
1.1. Generation of inducible shRNA mediated knockout lines 20
1.2. Testing the requirement of PDH by knockdown experiments 23
1.3. Role of other mitochondrial genes found in complex with CHD7, in 24
4
NCC formation like TIMM- 8, 13 using TIMM 9 and TSH as control
II. Assessing the role of PDH complex on NCC formation using
phosphorylation dependent inactivation in nuclear compartment
27
2.1. Confirmation of nuclear localized expression of NLS-PDK1 construct 30
2.2. Generation of GFP-NLS-PDK1 overexpression and control GFP line 31
2.3. NCC differentiation defect in NLS-PDK1 overexpression line 31
DISCUSSION 33
FUTURE WORK 36
REFERENCES 37

5
ABSTRACT:
Pyruvate dehydrogenase (PDH) complex, a major mitochondrial protein, is a complex of three
enzymatic subunits that convert pyruvate into acetyl CoA in a process called pyruvate
decarboxylation. Recent studies show that intact and functional PDH complex can translocate to
the nucleus in some cells and provide Acetyl-CoA substrate for histone acetylation (Sutendra et
al). Based on previous discoveries made by our lab, CHD7, an ATP dependent chromatin
remodeler was found to biochemically associate with all members of the PDH complex as well
as translocases of the inner membranes (TIM) in nuclear extracts of human neural crest cells
(hNCC). Moreover, abundant PDH complex was detected in the nuclei of developing neural
crest cells. Our lab and others, also showed that the disruption of CHD7 in Xenopus, Zebrafish
and hNCC blocked formation of multipotent migratory neural crest cells resulting in defects that
mimic CHARGE syndrome, a disease caused by mutations in CHD7. Here we hypothesize that
CHD7 associated PDH complex provides substrate for hyper-acetylation and rapid
activation of neural crest specific CHD7 target genes. Thus, I headed to explore the functions
of nuclear PDH complex and associated proteins in hNCC formation from embryonic stem cells
by downregulating the expression of PDH complex/ TIMMS using inducible shRNA constructs.
Further, to selectively knockdown the function of PDH complex in the nucleus, we utilized the
well-known ability of pyruvate dehydrogenase kinase (PDK1) to phosphorylate and inactive
PDH and the NCCs that developed showed disruptions, which mimics the phenotypes observed
in CHARGE patient derived iPSC.

6
INTRODUCTION:
One major cause of infant mortality is craniofacial disorders, that are devastating to children and
parents. According to statistics, CHARGE syndrome occurs in every 9-10,000 of the babies born
worldwide who show defects in cleft lip, cleft palate, small or absent facial and skull bones and
improperly formed nose, eyes, ears, and teeth. There is so far no potential solution for cure of
CHARGE but treatment methods like surgery and dental care can ameliorate various defects but
to a limited extent at great cost over many years. Treating the craniofacial abnormalities often
vary and are rarely fully corrective. Treatment of developmental defects requires remedies for
tissue repair and regeneration so as to eliminate the devastating consequences of head and facial
birth defects. Developing an effective remedy requires profound understanding of the normal
events that control craniofacial development during embryogenesis (Trainor PA et al., 2010).
Craniofacial morphogenesis, a complex process of embryogenesis leads to the vertebrate head
and face formation. The neural plate consists of three distinct tissue layers known as ectoderm
(outside), mesoderm (middle), and endoderm (inside), which folds to form the neural tube during
neurulation. Subsequently, neural crest cells from the dorsal part of the neural tube undergo
an epithelial to mesenchymal transition, delaminating from the neuroepithelium and migrating to
the periphery where they differentiate into varied cell types (Huang et al., 2011). Neural crest
cells are derived from the neural ectoderm and migrate over long distances and forms cartilage,
bone, connective tissue, sensory neurons, glia, and pigments cells and many other cell types and
tissues. These neural crest cells thus contribute to the formation of facial structures and when
defective results in many craniofacial abnormalities.
7

Figure 1: The neural plate folding to form the neural tube. The NCCs arise from the dorsal part
of the neural tube and undergo Epithelial-Mesenchymal Transition. The NCC then delaminate
and migrate to form different tissues of the body (Trainor PA et al., 2010)

CHD7 and CHARGE Syndrome:
Heterozygous loss of CHD7, leads to a congenital malformation disorder CHARGE syndrome,
with craniofacial defects such as choanal atresia or cleft lip or palate, ocular colobomas,
cardiovascular malformations, retardation of growth, ear anomalies, and deafness (Bergman et
al. 2011; Jongmans et al. 2006; Lalani et al. 2006; Morgan et al. 1993; Sanlaville and Verloes
2007). The CHD7 protein, is a highly conserved ATP dependent transcriptional regulatory
protein and plays a significant pleiotropic role in early embryonic development by controlling
gene expression in a variety of tissues and also in adults (Bouazoune K et al., 2012, Layman WS
et al., 2010 and Janssen N et al., 2012). The CHD7 expression in mouse embryos (Lalani et al.,
8
2006) showed the variable levels of CHD7 expression in different tissue types and is highly
expressed in the tissues which are majorly impaired in CHARGE syndrome (Figure 2).

Initially it was postulated that CHARGE syndrome was due to improper neural crest cell
development (Siebert et al., 1985) and confirmed that CHD7, regulates the formation of
multipotent migratory neural crest (NC), a transient cell population that is initially ectodermal in
origin but undergoes a major transcriptional reprogramming event to become mesenchymal and
Figure 2: Insitu hybridization of
mouse embryo and sagittal sections
showing the expression of CHD7 in
craniofacial region (A), cardiac
outflow tract (OFT) and atria (B),
facio-acoustic, hindbrain (HB),
forebrain (FB), mandibular
component of the first branchial arch
(BA), and otic vesicle (OV) (C), optic
stalk/optic vesicle (OPV) and
olfactory pit (OP) (D and F) at day
10.5 (Lalani et al., 2006)
9
acquire much broader differentiation and migratory properties, giving rise to craniofacial bones
and cartilages, the peripheral nervous system, pigmentation and cardiac structures (Sauka-
Spengier T et al., 2008 and Dupin E et al., 2006). In humans and Xenopus, CHD7 regulates the
neural crest development by turning on transcription factors (Slug, Twist and Sox9) necessary
for the formation of early migrating NCCs (Bajpai et al., 2009).
CHD7 is a multi-domain ATP dependent chromatin remodeling protein without any known
sequence specificity for binding DNA. The two chromo domains at its N-terminus, however
were found to bind histone 3 that is methylated at lysine 4( H3K4methyl) in vitro. In addition
CHD7 was found to be closely associated with regions of H3K4 methylation in vivo that
represent active or open chromatin signatures. Although CHD7 binding sites vary between cell
types as described in Mouse Embryonic stem cell, Neural progenitor cell lines and
neuroblastoma cells, in each case they overlap with the transcriptionally active chromatin regions
that are DNase hypersensitive with H3K4 methylation marks. (Schnetz, Michael P et al., 2009).
Similarly, transiently activated neural crest specific developmental genes and their enhancers
were identified as CHD7 target in human neural crest cells but not related cells like neural
precursors or pluripotent stem cells.

10
Figure 3: The CHD7 binding sites overlap with the transcriptionally active histone marks/ DNase
hypersensitive or open chromatin regions (Schnetz, Michael P et al., 2009).

Figure 4: In mouse Embryonic stem cell and Neural progenitor lineage, the CHD7 binding sites
overlap with regions of H3K4 mono methylation that marks the enhancer regulatory elements.
Our lab made a surprising discovery of the physical association of CHD7 with Pyruvate
dehydrogenase complex, a major mitochondrial protein, inside the nucleus of human neural crest
cells as well as two specific translocases of inner mitochondrial membrane- TIMM 8 and 13, by
co-immunoprecipitation followed by mass spectrometry. Thus we headed to explore if the
nuclear pyruvate dehydrogenase complex has a functional role in neural crest development
especially in regulating neural crest specific genes downstream of CHD7, that are specifically
affected in CHARGE patients.

11
Protein ID: Mascot score
CHD7: 10,040
PDH-E2p: 760
PDH-E1b: 528
PDH-E2: 439
PDH-E1a: 276
PDC-E2bp: 271
PDC-E3: 119

Figure 5: Mass spectrometry data showing the physical interaction of CHD7 with all the
subunits of PDH in nuclei of human neural crest cells (Ruchi Bajpai).

Pyruvate dehydrogenase complex:
Pyruvate dehydrogenase complex (PDH), abundantly present in the mitochondrial matrix, is a
complex of three enzymes E1 pyruvate dehydrogenase, E2 dihydrolipoamide acetyltransferase
and E3 dihydrolipoamide dehydrogenase that converts pyruvate to acetyl-coA by a process
called pyruvate decarboxylation. Acetyl-CoA is a central molecule in metabolism with a high
energy thioester linkage that serves a s an excellent donor of acetate moiety. It may then be used
in the citric acid cycle to carry out cellular respiration, and this complex links the glycolysis
metabolic pathway to the citric acid cycle. Sutendra et al., shows proof of concept that functional
PDC generates acetyl-CoA in the nucleus independent of mitochondria. It is observed that all
components of PDC translocate from mitochondria to the nucleus in response to multiple signals
and this functional nuclear PDH generates acetyl CoA, important for histone acetylation for S
phase progression. However, these conclusions were drawn from experiments where pyruvate
dehydrogenase was knocked down in the entire cell including mitochondria or in isolated nuclei.
With energy status of the cell compromised upon PDH knockdown, it is difficult to assess
whether the effect on histone acetylation is directly due to lack of acetyl CoA supplied by PDH
or not.
12

Figure 6: Co staining of PDH-E1 (green), mitotracker mitochondrial marker (red) and nuclear
marker DAPI (blue) or Histone3 (purple) showing the presence of PDH-E1 in the nucleus of
Primary fibroblasts (A), small airway epithelial cells (SAECs) (B), and A549 cells (C).
Enzymatic activity of PDH complex is regulated by the abundance of substrates and products of
the reaction. It is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA
(CoASH) and NAD
+
. Acetyl-CoA is also however not freely diffusible across nuclear membrane
and this small molecule cannot be used for inactivating nuclear PDH complex.
Phosphorylation state of the PDH complex also regulates its activity. The PDH complex is
phosphorylated by specific kinases (PDK1–PDK4) that inhibit its activity and lead to decreased
oxidation of pyruvate. To reverse their inhibitory effect, the phosphate groups on PDH complex
are removed by a specific phosphatase (PDH phosphatase, PDP). PDH-kinases are activated by
NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca
2+
and Mg
2+
. PDP, in
contrast, is activated by Mg
2+
and Ca
2+
. All isoforms of pyruvate dehydrogenase kinase have a
classical mitochondrial localization signal at the N terminus and are responsible for the
mitochondrial localization of the protein. Low levels of the PDH-kinase is also found in the
cytoplasm but these kinases are excluded from the nucleus. (Sutendra et al., 2014). PDH kinases
regulates the activity of the mammalian PDH complex by phosphorylation of three specific
serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the α subunit of the pyruvate
13
dehydrogenase (E1) component at different rates. It was shown that out of the four isoforms,
PDK1 was capable of phosphorylation of all three sites of the Pyruvate dehydrogenase complex
(LG Korotchkina et al., 2001).
7A 7B
Figure 7: (A) A549 and (B) 786-O cells stained for PDKII and PDKI showing the absence of
PDK in the nucleus (Sutendra et al., 2014).

Hence to study the role of nuclear PDH complex by specifically inactivating it in the nucleus, we
propose to use the inhibitory activity of the PDK1 protein tagged to a nuclear localization signal
so that the activity of the mitochondrial PDH is not altered.

14
Figure 8: The inactivation strategy of nuclear PDH complex by phosphorylation using a nuclear
localized PDK1 with GFP reporter. The mitochondrial PDH complex remains functional and
active.
So in order to study the role of the nuclear PDH in the neural crest cell development, we decided
to inactivate the function of PDH complex using a lentivirus based inducible NLS-PDK1
construct.

The Human Stem cell differentiation model:
An in vitro model of human neural crest cell was and extensively developed and characterized in
our lab. The hESCs when differentiated with collagenase were induced to form neuro ectodermal
Pyruvate
Coenzyme

A
NAD
+

Acetyl-‐CoA
CO
2

NADH;
H
+

PDH

phosphatase

Pyruvate

Dehydrogenase

Kinase-‐1
Inactive

PDH

complex
ATP
ADP
Pi
P
P
P
P
P
P
P
P
NLS
Active

PDH

complex
mitochondria
15
spheres like rosettes, which started attaching on day 6 and gave rise to stellate-morphology
shaped migratory cells. These migratory cells where characterized to be Neural crest cells as they
express early NC genes (SOX9, AP2α, p75) which was further confirmed by immuno stainings.

Figure 9: (A) Model of hNCLC derivation. hESCs were differentiated in into neuroepithelial
spheres, which spontaneously attach on day 6 and form migrating neural crest cells. The
migratory cells were then immuno stained for early characteristic NC markers like AP2-α,
nestin, surface p75 and 30% of NCCs stained for HNK1, as above.

16
MATERIALS AND METHODS:
Tissue culture growth conditions
All tissue culture cells were grown in a Sanyo CO2 incubator at 37°C with a [CO2] of 5.0%. We
used complete mTeSR medium (Stem Cell Technologies) with 5X supplement (Stem cell
Technologies) to culture the ESC in a serum free-environment (Vallier 2011). ESC lines were
maintained by passaging using accutase (Stem Cell Technologies) according to the method
described in Bajpai et al., 2008. ESCs were differentiated into neural crest cells by collecting
ESC by treating them with collagenase and then culturing small clusters in media composed of
1:1 ratio of DMEM/F12 neurobasal medium with 0.5%N2 (Life technologies), 0.5%B27(gem21)
supplement (Gemini), 5 ug/ml insulin, 20 ng/ml bFGF, and 20 ng/ml EGF (Bajpai et al., 2009).
Media was changed the next day, and then added as needed.
Embryonic stem cell (ESC) lines:
The base cell line used in these studies was H9 (WA09 from Wicell) human embryonic stem
cells (hESC) in accordance with SCRO approved protocols. Using lentiviral mediated infection
described in Bajpai 2011 with minor modifications, we created GFP reporter lines SIN18 PGK-
GFP as positive control, inducible Tz NLS-PDK-GFP line and inducible shRNA lines to knock
down PDH, TIMM8, TIMM9 and TIMM13 as well as a non targeting shRNA construct as
negative control referred hereafter as Tsh. The lentiviruses were generated by transfecting HEK-
293T cells with three plasmids that contained lentiviral packaging components plus the transfer
vector with the enhancer-reporter sequence. Lentivirus was produced using second generation
packaging system. This system utilizes the replication incompetent viral vector and packaging
proteins (VSVG, gag-pol, and CMVΔR8.74).
17

Figure 10: Table showing the shRNA constructs that were used for creating knockout lines.
Lines with shRNA were created with a transfer plasmid containing a doxycycline inducible
shRNA of the desired gene, PDH, TIMM 13, TIMM 9, TIMM 8 and control Tsh located in the
3’UTR of RFP reporter. Thus, the expression of RFP is a direct measure of the shRNA being
produced. The shRNA construct also contains an independent puromycin selectable marker
driven by a constitutive Ubiquitin C (UbC) promoter. In a separate construct, the RFP-shRNA
segment has been replaced by NLS-PDK-GFP fusion protein. The puromycin resistance gene
driven by a second Ubiquitin C promoter is included for selection of pluripotent ESC infected
with NLS-PDK-GFP. The human Sin18 PGK-GFP positive control vector has GFP expression
driven by a constitutive phosphoglucokinase promoter.
Transfection of the HEK-293T cells was done using Polyethylenimine (PEI) according to
methods described in Boussif et al., 1995. Media was changed to serum free Ultraculture
medium( Lonza) supplemented with 2x glutamine and penicillin-streptomycin on the second day.
Virus was secreted into the medium for the next 36-48 hrs. The harvested virus was spun down
18
at 2400 rpm for 10 min to remove debri then concentrated by ultracentrifugation in a Beckman
swinging bucket rotor (SW28) at 16,000 rpm overnight. The supernatant was discarded and the
concentrated virus was resuspended by constant shaking at 4 degrees for at least 4 hrs overnight.
Human ESCs were infected with lentivirus in suspension for 30 mins by incubating harvested
clusters of cells in minimal volume (100-300ul) containing matrigel diluted 1:90 in mTESR
supplemented with 5 ug/ml polybrene and 75-90ul concentrated lentivirus. The cells with virus
were transferred to matrigel coated 6- well dishes in 2 ml mTESR. Media was changed every day
thereafter. The clusters attached to the dish and grew as colonies. In hESC infection the region
between the LTRs of the transfer plasmid is inserted into the hESC genome, creating a stable line
with the insertion described above. hESC colonies were passaged every 5-6 days. During the first
passage the colonies were dissociated in very small clusters (3-30 cells) and replated in media
containing 2ug/ml puromycin. Only infected cells are resistant to puromycin and form
independent colonies. Single colonies from different wells were treated as independent clonal
populations. In addition, for enrichment of RFP tagged shRNA infected and doxycycline
responsive pool populations or clonally infected colonies of hES cells were treated with 2ug/ml
doxycycline for 24 hrs followed by FACS sorting RFP positive population. The positive cells
were re-aggregated in mTESR supplemented with matrigel in suspension for 30mins and then
expanded on matrigel coated wells in the absence of doxycycline. Enriched cell populations were
either frozen in liquid nitrogen to generate stocks or used for NCC differentiation assay with or
without Doxycyclin.

19
Imaging
Images of the tissue culture samples were taken with a fluorescent microscope (Leica DMI3000
B) and a confocal microscope (Broadband Confocal Leica TCS SP5 II). All photos were
analyzed and assembled on Leica LAS AF Software.
FACs sorting
FACs sorting was done with BD FACS AriaTM cell sorter (BD Biosciences) at the University of
Southern California Flow Cytometry Core Facility. Excitation lines used are 488nm and 561nm.
Intensity of laser determined by negative control, negative control used for FACs was always
cells at the same stage in differentiation with no fluorescence markers.
Immunoprecipitation and Western Blotting:
The nuclear proteins (overexpressed nuclear PDK-GFP line) were extracted using Dignam A
(10mM Hepes/KOH pH 7.9, 1.5mM MgCl2, 10mM KCl) and high salt extraction buffers
(100mM Tris HCl pH 8.0, 300mM NaCl, 0.1% NP40, 10% Glycerol, 1mM EDTA, 1mM
EGTA). The whole cell proteins (PGK-GFP) were extracted using RIPA buffer (50mM Tris
HCl, 1% NP40, 0.5% Na deoxycholate, 0.1% SDS and 150mM NaCl). Immunoprecipitation was
done using Dynal Beads A and G. Ideally the protein is incubated with the primary antibody
right after extraction for 2 hrs. Dynal A+G beads washed with 150mM salt buffer are then added
to the mixture and incubated for an additional hour followed by washing of the bound proteins.
Sometimes protein extract is pre-incubated with the beads to remove non-specific binding (20
minutes). Beads can also be incubated with block buffer for 20 minutes to saturate non-specific
sticky surfaces on the beads. The bound proteins were then washed with 150mM salt buffer and
20
eluted by 4X SDS loading buffer on magnetic stand. The eluted protein was denatured at 95°C
for 5minutes and separated from the beads.
The eluted protein was then loaded on 10% SDS PAGE and then transferred on to the
nitrocellulose membrane. The membrane was then probed with anti-GFP antibody (Invitrogen)
in a 1:1000 ratio in 1X Roche Blocking buffer for overnight at 4°C. The membrane was then
washed and probed with the corresponding secondary antibody (Goat anti Rabbit) in 1:20,000
ratio in 1X TBST.
RESULTS:
Section I: Assessing role of PDH and associated protein complexes on NCC formation using
inducible knockdown technology
1.1. Generation of inducible shRNA mediated knockout lines:
The inducible shRNA mediated knockout lines were generated with the help of Tet-ON lentiviral
constructs. The pentagons represent the LTR regions, flanking the insertion (Figure 8). The
insertion construct contains tet- inducible promoter driving expression of RFP together with
shRNA targeting one of the following genes-
TIMM8
, TIMM9, TIMM13, PDH and Tsh (non
targeting control). The constitutive promoter UbC drives expression of rTA3, a third generation
reverse trans-activator that can bind tet-operator (tetO) only in the presence of tetracyclin or its
analog Doxycyclin and drive expression of RFP from the minimal promoter. Thus, RFP and
shRNA are not expressed in the absence of Doxycyclin. Once shRNA is produced it triggers the
degradation of the specifically targeted transcript. In addition, the shRNA is flanked by
microRNA, miR 5’ and miR 3’ sequences, which provide an additional mechanism of
microRNA mediated decrease in translation of the target gene. This dual knockdown strategy has
21
proven to be extremely successful in effectively knocking down genes with even single copy
insertions of the construct (Ellidge and Hannon). The level of knockdown is expected to be
directly proportional to RFP intensity, as the shRNA is within the 3’UTR of RFP and one
molecule of shRNA is generated for every copy of RFP transcript. A puromycin resistance gene
is used for independent selection of the ESC with the lentiviral insertion.

Figure 11: Lentiviral construct insertion used to infect ESC and generate different shRNA H9
lines.
Virus was generated and used to infect karyotypically normal and mycoplasma free H9 cells
obtained from Loring lab at The Scripps research institute, A Jolla, CA. Puromycin selection
resulted in <10% survival of infected cells while all control, uninfected cells were killed. Since
low infection rates preclude multiple infections we expect that the surviving colonies have single
copy infections.

miRNA
flanks
6x tet
rTA3 Puro
UbC
Promoter
shRNA
22

Figure 12: Lentiviral mediated transduction method used to generate hESC knockdown reporter
lines. Example of positive control lentivirus (PGK-GFP is shown) (Bajpai et al., 2011). 1.Co-
transfection of lentiviral assembly plasmids into HEK-293T cells. Plasmids contain lentiviral
production components plus the transfer vector with the enhancer- reporter sequence. HEK-293t
cells transduce the lentivirus. 2.Lentivirus is released into the medium, and then the medium is
collected. From which the virus is concentrated. 3.Infection of ESC with the lentivirus (75µl of
PGK GFP and 110µl of GFP-NLS-PDK1). The insertion region of the transfer vector, is flanked
by LTR sequences, is inserted into the ESC genome. 4.The stable ESC with the insertion is
created and can be selected for using antibiotic resistance and FACs sorting.
23
1.2. Testing the requirement of PDH by knockdown experiments:
In order to test the importance of cellular PDH, we knocked down PDHA1 subunit in hES using
the shRNA mediated lentiviral approach. It was seen that knockdown of PDH in ES cells
resulted in complete cell death, thus unable to survive for longer than 2 days or differentiate in
the presence of Doxycyclin. Further infected cells selected with puromycin were able to
upregulate RFP-tagged shRNA transiently, but these RFP positive cells were unable to survive in
differentiating hNCC cultures or in 293T.

B

24
Figure 13: (A) Transfection of shPDH in 293T cells and observed cell death in ES cells infected
with shPDHA1 FACS analysis of shPDHA1 cells. (B) Panel 1: negative control (uninfected cells
or infected cells without doxycycline were comparable with no leaky RFP expression). Panel
2,3: Cells in differentiation with doxycycline showed RFP expression in >50% of the cells within
24-36 hrs. However rare or no RFP positive cells were detected after 4 days in doxycycline (cells
analyzed at 4, 6, 10 days; data not shown) (Jennifer Oki)
1.3. Role of other mitochondrial genes found in complex with CHD7, in NCC formation
like TIMM- 8, 13 using TIMM 9 and TSH as control
Multiple shRNA knockdown lines of other CHD7 associated proteins were developed in ES cells
by lentiviral mediated knockdown. When the lentivirus gets integrated into the ES cell genome,
RFP is expressed in the presence of doxycycline. The shRNA infected ESC’s were treated with
collagenase and put into a neural differentiation media, in which they differentiated to neuro
ectodermal spheres and migrating neural crest cells. Once in the media, neuroectodermal spheres
start to form and grow, these cells are analogous to cells in the neural tube of an embryo. Around
day 7 or 8 the spheres start attaching to the dish and neural crest cells start to migrate from them.
It was observed that (on day 8), upon differentiation the shTIMM8 and shTIMM13 did not inhibit
the formation of neural crest but their multipotent migratory property (Figure 14) whereas
control shTIMM 9 and non targeting Tsh had no effect (Figure 12).

25

Figure14: (A) ES cells with TIMM9 knockdown showing proper development of multipotent
migrating neural crest cells (B) when differentiated. Control Tsh lines, also show proper
development of neural crest cells (C). Both RFP positive (solid white line) and RFP negative
(dotted white line) NCCs migrate to the same extent in TIMM9 and Tsh control lines.
26

Figure15: Generated ESC with TIMM8 (A) and TIMM13 (B) knockdown by lentivirus. Proper
rosette formation and initial differentiation of neuro ectodermal spheres TIMM8 (C) and
TIMM13 (D) knockdown lines. Migration of neural crest cells getting affected in RFP positive
NCCs of TIMM8 (E) and TIMM9 (F). (White arrows) showing the difference in the distance of
migration of neural crest cells with the shRNA knockdown NCCs (RFP positive) (indicated by
solid white line) and RFP negative NCCs (dotted white lines).

27
To further validate the knockdown of the respective gene and other NCC specific genes, we
subjected the shRNA knockdown hNCC lines to FACS sort on day 10, and the sorted cells were
collected in Trizol and frozen in -30°C for future RNA seq analysis. (Figure 16).

Figure 16: FACS sorting data showing the number for RFP positive cells, collected on day 10
after differentiation, under induced conditions, Y-axis represents the intensity of RFP. Panels
Tsh (Positive control), TP1 and TIMM9 showed high RFP expression, whereas panels TIMM13
and TIM8 show reduced expression of RFP, and panels PDH1α and normal hNCC (negative
control) show no expression of RFP.
Section II: Assessing the role of PDH complex on NCC formation using phosphorylation
dependent inactivation in nuclear compartment
The pyruvate dehydrogenase kinase was observed to be absent in the nuclei of A594 cells
(Sutendra et al.,2014). The nuclear pyruvate dehydrogenase kinase was overexpressed using
28
lentivirus mediated method to knockdown PDH by phosphorylation.

Figure 17: Lentiviral construct insertion used to infect ESC and generate GFP-NLS-PDK1 H9
lines
The pentagons represent the LTR regions, flanking the insertion. To observe activation of the
nuclear PDK1 in human tissue culture, lentiviral mediated transduction on ESCs inserted the
nuclear PDK1 tagged with GFP into the ESC genome. Thus, creating cell line H9- NLS PDK1
GFP. The insertion construct contains a doxycycline inducible tet operator system and
puromycin resistance for selection of clones.
2.1. Confirmation of nuclear localized expression of GFP-NLS-PDK1 construct
293T cells, were transfected with the GFP-NLS-PDK1 and PGK GFP (positive control) whole
cell lysates prepared by RIPA buffer. Western blot of the whole cell lysates show the fusion
protein is expressed and then immunofluorescent stainings with mitotracker and DAPI show that
the GFP-NLS-PDK1 is primarily nuclear localized with low levels detected in the cytoplasm.
However, GFP-NLS-PDK1 appears excluded from the mitochondria (as expected) (Figure 18).

29

30

Figure18: 293T cells transfected with GFP-NLS-PDK1 (a) and PGK GFP (b) (without packaging
plasmids) showing the expression of GFP from which whole cell lysates were prepared. (C)
Immunofluorescent staining with mitotracker and DAPI confirming the presence of GFP-NLS-
PDK1 in the nucleus. (D) Western blot with GFP antibody (Invitrogen) (Rabbit) showing the
presence of GFP in the whole cell lysate of PGK GFP (28kDa) and Fusion protein GFP-NLS-
PDK1 (72kDa). (E) Immunoprecipitation with GFP antibody (Invitrogen) and western blot
confirming the presence of GFP in 293T transfected with PGK GFP.
2.2. Generation of GFP-NLS-PDK1 overexpression line and control GFP line
In order to explore the role of nuclear PDH, we inactivated it by expressing it inhibitory kinase,
Pyruvate dehydrogenase kinase with a nuclear localization signal and GFP reporter tag. The
GFP-NLS-PDK1 and PGK GFP were overexpressed in the ES cells by the lentiviral mediated
transduction method as described earlier (Figure 12). In the presence of doxycycline GFP the
NLS PDK1 gene gets integrated into the genome and GFP is expressed, whereas in PGK GFP,
the GFP expression is driven by phosphoglucokinase promoter and is always on (Figure 19).
31

Figure19: 293T transfection of GFP NLS PDK (A) and PGK GFP (B) in 293T cells.
Development of ES cell lines with overexpressing GFP-NLS-PDK1 in +Dox (C) and PGK GFP.
2.3. NCC differentiation defect in NLS-PDK1 overexpression line
The infected ES lines were treated with collagenase and put into a neural differentiation media,
in which they differentiated to neuro ectodermal spheres and migrating neural crest cells. In
GFP-NLS-PDK1, the migration of neural crest cells was inhibited under induced conditions
(+Doxycycline), whereas unaffected in non induced conditions, showing that inhibiting the
activity of nuclear Pyruvate dehydrogenase complex by nuclear PDK1 has an effect on the
formation of multipotent migratory neural crest cells. In constitutive GFP expressing control
lines, addition of doxycycline did not cause any significant difference in NCC formation and
migration (data not shown).

32

Figure 20: (A) The neural spheres of GFP-NLS-PDK1 show proper formation of multifunctional
migratory neural crest cells (dotted lines) in non induced (-Dox) conditions and (B) impaired
formation of neural crest cells under induced (+Dox) conditions, showing no (solid white line)
and very little migration of NCCs (dotted orange lines).
On day 7, the neuro ectodermal spheres were transferred to several 12 well dishes and allowed to
adhere and later fixed on day 10 with 4% PFA. The spheres were then counted for improper
development of neural crest cells. Three migration patterns were observed. The spheres from
which the NCCs migrated extensively over long distance was termed “normal”, whereas the
phenotypic spheres from which there was no migrating NCCs was termed “strong” and the ones
which gave rise to NCCs but couldn’t migrate over long distances and appeared stuck was
termed “mild”. In GFP-NLS-PDK1 overexpressing hNCCs (+Dox) about 46% of the spheres
showed normal migration of NCCs, whereas there was a strong phenotype of 29% and a mild
phenotypic expression of 25% (Figure 21).
33

Figure 21: Bar 1, showing the total number of neuro ectodermal spheres in normal (blue), mild
(orange) and strong (red) phenotypes. Bar 2 represents the neuro ectodermal spheres in induced
(+Dox) conditions.
DISCUSSION:
CHD7 is a ATP dependent chromatin remodeling protein that is essential for the formation of
multipotent migrating neural crest cells and regulates genes involved in the development of
neural crest cells (Randall et al., 2009, Bajpai et al., 2010, Schulz Y et al., 2014). Knockdown of
CHD7 affects the formation of multipotent migrating neural crest cells in Xenopus laevis (Bajpai
et al., 2010).
PDC deficiency, an inborn error, impairs the mitochondrial energetics that may lead to
neuropathology of Leigh syndrome, with spongy degeneration in the thalamus, basal ganglia and
brain stem (Robinson BH et al., 2001 and Leigh et al., 1951). And so far there are no proven
methods for treating inborn PDH deficiency (Carolyn Ojano-Dirain et al., 2010).
78%
46%
16%
25%
6%
29%
0%
20%
40%
60%
80%
100%
120%
No
Dox Plus
Dox
strong
Mild
Normal
34

Children born with PDH deficiency show defects in muscle tone, cognitive and developmental
delays which is in close resemblance and a subset of the CHARGE defects. Thus the mild
spectra of defects observed in CHARGE patients who do not posses CHD7 mutations could be
possibly due to the nuclear PDH deficiency or the other associated proteins and their loss of
function. While CHD7 may have a critical function in only a subset of cells PDH complex is
universally present and expected to have a much broader effect on many organs and tissues.
Since the acetylation status of the various genes is an important hallmark for turning on the
neural crest specific genes, the availability of the substrate acetyl-CoA plays a major role. Thus
reduced availability of glucose during fetal development can also cause a major impact on the
neural crest cell development.
Complete deficiency of PDHE1α is a lethal defect for the developing embryos (Johnson MT et
al., 2001 and Sidhu S et al., 2008). Therefore, conditional knockout strategies offer a potential
alternative. Thus the inducible hESC knockout model could be a potential one to study the role
of nuclear PDH complex in neural crest development.

35
Figure 22: Knockdown of CHD7 in Xenopus, shows diminished expression levels of Sox9, early
neural crest marker, and two critical neural crest and EMT regulators Twist and Slug (Bajpai et
al., 2010).
Like CHD7 knockdown, it is also observed that inactivation of nuclear PDH by NLS-PDK GFP
in Zebra fish embryos affects the formation of multipotent migratory neural crest cells (Ruchi
Bajpai). Sox10, a multipotent neural crest cell marker shows that zebrafish embryos with GFP-
NLS-PDK1 mRNA, affects the ability to form multipotent migratory neural crest cells.

Figure 23: (A) Zebra fish embryos injected with control mRNA showing normal migration of
neural crest cells (B) GFP-NLS-PDK1 mRNA shows the expression of Sox10 in newly
generated neural crest cells but a defect in their ability to migrate ( Ruchi Bajpai ).
Our preliminary data suggests that nuclear PDH complex, TIMM8 and TIMM13 affects the
development and migration of human NCCs , whereas TIMM 9 and Tsh did not. If the
corressponding defect is due to their role in translocating PDH complex to the nucleus or due to
their association with CHD7 and the activation of downstream neural crest specific genes it can
be validated only after testing the degree of knockdown, by RT-qPCR. Additional methods to
36
validate the specific effect of the shRNA on the targeted gene is be by western blot to check if
there is a specific decrease in the targeted protein or by RNA sequencing to determine the
decrease in the mRNA level of the corresponding gene.
Even when an shRNA successfully targets the gene of interest resulting in effective knockdown,
shRNA’s may often possess additional off target effects, which leads to silencing of genes apart
from our target gene. Off-target effects of the shRNA giving rise to our observed phenotype can
be ruled out by (i) using several independent clones of the same shRNAs (ii) identifying at least
two independent shRNA sequences targeting the same gene that produce a comparable
phenotype (iiii) independent knockdown of multiple components of a functional complex
resulting in comparable phenotypes .
Our hypothesis of the association of CHD7 with the nuclear PDH complex provides substrate for
hyper-acetylation and rapid activation of neural crest specific CHD7 target genes can be verified
from the histone acetylation status of neural crest specific enhancers and genes.
CHARGE patients show a broad range of mutations and phenotypic spectra. This might be as a
result of severe mutations in CHD7 which results in strong developmental defects or could
possibly be due to the defects or variations in other CHD7 associated proteins and their
mutations. If the nuclear PDH proves to provide substrate for the activation of neural crest
specific genes and enhancers, then exogenously providing its substrate can be a potential method
to treat developmental defects like CHARGE, PDH-deficiency and other developmental
anomalies occurring during birth.
FUTURE WORK:
In order to strengthen these preliminary findings a few more additional experiments are required.
37
Immunofluorescent stainings of early NCC specific markers on the various knockdown lines of
hNCCs can verify the charecteristic neural crest cells and the migrational ability of the shRNA
neural crest cells.
RNA sequencing to test the degree of knockdown of the various shRNA and GFP-NLS-PDK1
lines. It can confirm the decrease in the mRNA levels of various knockdown lines of the
respective gene and thus reduced protein synthesis. RNA seq to show the increase in the mRNA
levels of PDK1 and the corressponding decrease in the PDH and CHD7 levels and various neural
crest specific genes downstream of CHD7, thus confirming its association with CHD7 dependent
mechanism.
To confirm the inactivation of PDH in the nucleus and not in the mitochondria of the GFP-NLS-
PDK1 lines. PDK1 inactivates PDH by phosporylating it at three serine specific sites. By
phospho serine specific antibodies, the functional activity of the PDH in the nuclei and
mitochondria can be determined. ATAC seq to determine the histone acetylation levels of the
various neural crest specific genes and ehancers during functionally active and inactive nuclear
PDH.
Inorder to confirm the colocalisation of CHD7 with the various subunits of nuclear PDH inside
the nucleus of human NCCs, immunostainings of PDH and CHD7 have to be done.
References:
1. Trainor PA., (2010). Craniofacial birth defects: The role of neural crest cells in the
etiology and pathogenesis Treacher Collins syndrome and the potential for prevention.
American journal of Medical genetics. 152A (12): 2984-2994.
38
2. Huang P et al., (2011). Induction of functional hepatocyte-like cells from mouse
fibroblasts by defined factors. Nature. 475: 386–389.
3. Bergman JE et al., (2011). Anosmia Predicts Hypogonadotropic Hypogonadism in
CHARGE Syndrome. Journal of Pediatrics. 158 (3): 474-479.
4. Jongmans, MC. et al., (2006). CHARGE syndrome: the phenotypic spectrum of
mutations in the CHD7 gene. Journal of Medical Genetics. 43: 306–314.
5. Lalani, SR et al., (2006). Spectrum of CHD7 Mutations in 110 Individuals with
CHARGE Syndrome and Genotype-Phenotype Correlation. American Journal of Human
Genetics; 78: 303–314.
6. Morgan, D. et al.,1993. Ear–nose–throat abnormalities in the CHARGE association.
Arch. Otolaryngol. Head Neck Surgeory. 119, 49–54
7. Sanlaville, D. and Verloes, A., (2007). CHARGE syndrome: an update. European Journal
of Human Genetics. 15, 389–399
8. Bouazoune, K. et al., 2012. Chromatin remodeling by the CHD7 protein is impaired by
mutations that cause human developmental disorders. Proceedings of the National
Academy of Sciences of the United States of America. 109 (47): 19238-43.
9. Layman, WS. et al., (2010). Chromodomain proteins in development: lessons from
CHARGE syndrome. Clinical Genetics. 78 (1): 11-20.
10. Korotchkina, LG. et al., (2001). Site specificity of four pyruvate dehydrogenase kinase
isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase.
Journal of Biological Chemistry. 276(40): 37223-9.
11. Vallier, L. et al,. (2011). Serum-free and feeder-free culture conditions for human
embryonic stem cells. Methods Mol Biol. 690 57-66. Print.
39
12. Bajpai et al., (2009). Molecular stages of rapid and uniform neuralization of human
embryonic stem cells. Cell Death Differ. 16(6): 807–825.
13. Bajpai, R. et al., (2011). Genetic Manipulation of Human Embryonic Stem Cells:
Lentivirus Vectors. Human Stem Cell Manual. 255-266. Print.
14. Boussif, O. et al., (1995). A versatile vector for gene and oligonucleotide transfer into
cells in culture and in vivo: Polyethylenimine. Proceedings of the National Academy of
Sciences of the United States of America. 92(16): 7297–7301.
15. B.H. Robinson et al., (1990). Defects in the E2 lipoyl transacetylase and the X-lipoyl
containing component of the pyruvate dehydrogenase complex in patients with lactic
acidemia, J. Clin. Invest. 85: 1821–1824.
16. Carolyn Ojano Dirain et al., (2010). An animal model of PDH deficiency using AAV8-
siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α. Molecular genetics
and metabolism. 101(2-3): 183-191.
17. Johnson, MT. et al., (2001). Inactivation of the murine pyruvate dehydrogenase (Pdha1)
gene and its effect on early embryonic development. Molecular genetics and metabolism.
74(3): 293-302. Janssen, N. et al., (2012). A novel classification system to predict the
pathogenic effects of CHD7 missense variants in CHARGE syndrome. Human Mutation.
33(8): 1251-60.
18. Sauka Spengler, T. et al., (2008). Evolution of the neural crest viewed from a gene
regulatory perspective. Genesis. 46(11): 673-82.
19. Duplin, E. et al., (2006). The contribution of the neural crest to the vertebrate body.
Advances in experimental Medical Biology. 589: 96-119
40
20. Bajpai, R. et al., (2010). CHD7 cooperates with PBAF to control multipotent neural crest
formation. Nature. 463 (7283): 958-62.
21. Schnetz, MP. et al., (2009). Genomic distribution of CHD7 on chromatin tracks H3K4
methylation patterns. Genome Research. 19(4): 590-601.
22. Sutendra et al., (2014). A nuclear pyruvate dehydrogenase complex is important for the
generation of acetyl-CoA and histone acetylation. Cell. 158(1): 84-97.
23. Johnson, MT. et al., (2001). Inactivation of the murine pyruvate dehydrogenase (Pdha1)
gene and its effect on early embryonic development. Molecular genetics and metabolism.
74(3): 293-302.
24. Johnson, MT. et al., (2001). Inactivation of the murine pyruvate dehydrogenase (Pdha1)
gene and its effect on early embryonic development. Molecular genetics and metabolism.
74(3): 293-302. Janssen, N. et al., (2012). A novel classification system to predict the
pathogenic effects of CHD7 missense variants in CHARGE syndrome. Human Mutation.
33(8): 1251-60.
25. Sauka Spengler, T. et al., (2008). Evolution of the neural crest viewed from a gene
regulatory perspective. Genesis. 46(11): 673-82.
26. Duplin, E. et al., (2006). The contribution of the neural crest to the vertebrate body.
Advances in experimental Medical Biology. 589: 96-119.
27. Bajpai, R. et al., (2010). CHD7 cooperates with PBAF to control multipotent neural crest
formation. Nature. 463 (7283): 958-62.
41

28. Schnetz, MP. et al., (2009). Genomic distribution of CHD7 on chromatin tracks H3K4
methylation patterns. Genome Research. 19(4): 590-601.
29. Sutendra et al., (2014). A nuclear pyruvate dehydrogenase complex is important for the
generation of acetyl-CoA and histone acetylation. Cell. 158(1): 84-97.
30. Johnson, MT. et al., (2001). Inactivation of the murine pyruvate dehydrogenase (Pdha1)
gene and its effect on early embryonic development. Molecular genetics and metabolism.
74(3): 293-302.

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Opposing function of chd7 and phf6 in zebrafish craniofacial development

PDF

Epigenetic checks and balances: PHF6 activity restricts neural crest migration

PDF

Chromatin remodeling factors Chd7 and Phf6 in craniofacial and heart development

PDF

Derivation, expansion and characterization of human hippocampal primordial cells from normal and diseased iPSCs

PDF

A novel role for post-migratory neural crest cells in cardiac outflow tract alignment: insights into the molecular basis of concomitant congenital cardiac and cleft malformations

PDF

Development of ECM for the preservation of adult mouse pancreatic islet function in vitro

PDF

The role of Hic-5 in glucocorticoid receptor binding to chromatin

PDF

Understanding the role of APP and DYRK1A in human brain pericytes

PDF

Lineage boundaries and cell migration in the patterning of the mammalian skull

PDF

A functional genomic approach based on shRNA-mediated gene silencing to delineate the role of NF-κB and cell death proteins in the survival and proliferation of KSHV associated primary effusion l...

PDF

Molecular mechanism of the recruitment of SWI/SNF chromatin remodeling complex and histone acetyltransferase to estrogen-responsive promoters

PDF

Genetics and the environment: evaluating the role of noncoding RNA in autism spectrum disorder

PDF

Retinoic acid and TGFβ signaling regulate cardiovascular development

PDF

Elucidating the role of neural crest specific Stat3 signaling in maintaining coronal suture patency during embryonic development

PDF

Mechanisms of nuclear translocation of pyruvate dehydrogenase and its effects on tumorigenesis of hepatocellular carcinoma…

PDF

Characterization of the retromer complex of proteins in gastric parietal cells

PDF

Mechanism of secretion and function of heat shock protein-90 (Hsp90) family genes

PDF

Identification and characterization of PR-Set7 and histone H4 lysine 20 methylation-associated proteins

PDF

The structure and function of membrane curving proteins on different membrane shapes and their regulation by post-translational modifications

PDF

The role of α-catlulin during tumor progression and early mouse development

Asset Metadata

Creator Meenakshi Sundaram, Uma (author)

Core Title Elucidating the functional role of CHD7 associated nuclear PDH complex and other associated proteins on neural crest development

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/26/2017

Defense Date 06/17/2016

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

Tag charge,chromatin remodeller,hyper acetylation,neural crest cells,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Bajpai, Ruchi (committee chair), Patel, Pragna (committee member), Xu, Jian (committee member)

Creator Email meenaksh@usc.edu,umasundaram12@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-287253

Unique identifier UC11281074

Identifier etd-MeenakshiS-4680.pdf (filename),usctheses-c40-287253 (legacy record id)

Legacy Identifier etd-MeenakshiS-4680.pdf

Dmrecord 287253

Document Type Thesis

Format application/pdf (imt)

Rights Meenakshi Sundaram, Uma

Type texts

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

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

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA