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Oct4 Links Multiple Epigenetic Pathways to the Pluripotency Network
Junjun Ding1, Huilei Xu2,#, Francesco Faiola1,#, Avi Ma’ayan2, and Jianlong Wang1,*
Supplemental Information
Contents
Supplemental Materials and Methods ............................................................................................. 2
Supplemental Figure Legends......................................................................................................... 5
Supplemental Table Legends .......................................................................................................... 9
Supplemental References .............................................................................................................. 10
1
Supplemental Materials and Methods
Generation of bioOct4 Expressing ESCs
To increase the level of functional
bio
Oct4 for efficient affinity purification, we setup the in vivo
biotinylation of Oct4 in ZHBTc4 cells (kindly provided by Dr. Austin Smith). In these cells,
endogenous Oct4 was deleted by sequential targeting. A doxycycline (dox)-suppressible
Oct4/Geo transgene was engineered into this cell line to maintain stem cell identity in the
absence of dox1. To adapt this cell line for in vivo biotinylation of Oct4, we replaced the doxOct4
transgene with the constitutive Oct4bio-BirA-IRES-EGFP by lentiviral infection in the presence
of dox (to suppress the
dox
Oct4 transgene). ESCs positive for EGFP were sorted by flow
cytometry and cultured for clones to grow. Positive clones were picked and expanded, and
expression of
bio
Oct4 and
dox
Oct4 was confirmed by western blotting (WB) using streptavidin–
HRP (GE Healthcare) and an anti-Oct4 antibody (Santa Cruz Biotechnology). Four ZHBTc4
clones expressing
bio
Oct4 were established. Standard procedures for ESC culture, western
blotting, and lentiviral infection were followed and have been described elsewhere2-3.
Nuclear Extract Preparation from ESCs
ZHBTc4 ESCs containing the biotinylated Oct4 and control ZHBTc4 cells were expanded to five
big square dishes (245 x 245 mm), washed with PBS, scraped off, and nuclear extracts were
prepared as previously described4. Briefly, the cells were extracted in Buffer A [10 mM HEPES
(pH 7.6), 1.5 mM MgCl2, 10 mM KCl] for 10 minutes to remove cytoplasmic proteins and
nuclear proteins were extracted by Buffer C [20 mM HEPES (pH 7.6), 25% glycerol (v/v), 0.42
M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA]. The salt concentration was decreased to 100 mM by
dialyzing to Buffer D [20 mM HEPES (pH 7.6), 0.2 mM EDTA, 1.5 mM MgCl 2, 100 mM KCl,
20% glycerol] at 4oC for 3 hours. The precipitates from dialysis were removed by centrifugation
2
at 25,000 x g for 30 minutes. The supernatants (freshly prepared nuclear extracts) were used for
affinity purification. The same procedure was applied for all ESC lines used in this study.
Size Exclusion Chromatography (Gel Filtration)
Size exclusion chromatography was performed in a DuoFlow BioLogic System according to the
manufacturer’s manual (BioRad). Briefly, nuclear extracts (10~20 mg) from ZO4B4 and J1
ESCs were applied to a S400 (HiPrep 16/60 Sephacryl) gel filtration column (Amersham
Biosciences), samples were eluted at 1 mL/min and continuously monitored with an online
detector at a wavelength of 280 nm. Fractions were collected, concentrated, and subjected to
SDS-PAGE followed by western blotting analyses with indicated antibodies. The S400 gel
filtration column was calibrated using the protein standards purchased from GE Healthcare
(cat#28-4038-41 LMW and cat#28-4038-42 HMW), and the relative sizes of the indicated
complexes were marked above the corresponding fractions. The void volume was determined
from blue dextran and the bed volume was determined from conductivity measurements per the
manufacturer’s instructions.
Coimmunoprecipitation and Immunoprecipitation
ESC lines expressing biotinylated candidate proteins such as Ring1B were established in the
BirA-expressing cells as previously described3. Co-immunoprecipitation using streptavidin (SA)
beads was performed as described for affinity purification and mass spectrometry in the main
text, except that the eluate from SA capture was used for western blot analyses.
For endogenous IP, nuclear extracts prepared from a 15-cm dish of parental J1 cells were diluted
with Buffer D (with 0.02% NP-40 and without KCl) to adjust the salt concentration to final 150
mM, and incubated O/N at 4C with 5 to 20 g of anti-Oct4 (Santa Cruz Biotechnologies sc5279) or mouse IgG (Millipore) antibodies. The immune-complexes were captured with 20 to 80
3
µL protein G agarose beads for 2 hours at 4C, and washed 4 times with Buffer D containing
0.02% NP-40. Immunoprecipitated proteins were then resolved by SDS-PAGE and analyzed by
western Blotting or subjected to LC-MS/MS for identification.
Bioinformatics Analyses
Hierarchical Clustering of Gene Expression Profiling of Oct4 Knockdown mESCs
The processed Oct4 shRNA knockdown gene expression dataset was downloaded from GEO
(GSE4679) and log2 transformed. Expression intensity for Oct4-associated proteins were
extracted (when multiple probe sets were identified, expression was averaged). A total of 191 out
of the 198 Oct4-associated proteins were available. Expression values are represented as foldchange on each day compared to day 0. Finally, complete-linkage hierarchical clustering was
performed along the columns and rows to obtain the final output using MATLAB (Natick, MA).
Hierarchical Clustering of Gene Expression Profiling of EB Differentiation
Time-course EB differentiation raw data was retrieved from GEO (GSE3749) and normalized
using RMA with quantile normalization for Affymetrix MOE430a. Expression data for Oct4associated proteins were extracted (when multiple probe sets were available, expression was
averaged). A total of 171 out of the 198 Oct4-associated proteins were identified. Expression
values are represented as fold-change on each time point compared to day 0. Next, completelinkage, hierarchical clustering was performed along the columns to obtain the final output.
Statistical Analysis for Functional Validation of Oct4 Interactome
Oct4, Sox2 and Nanog direct binding targets in mouse embryonic stem cells were extracted from
the ChEA database (http://amp.pharm.mssm.edu/lib/chea.jsp). Two-tailed Fisher’s test was
performed using the R package “exact2x2”.
4
Supplemental Figure Legends
Figure S1. Characterization of ZO4B4 ESCs.
(A) Quantitative presentation of the colony formation assay. ZHBTc4 and ZOB4 ESCs were
cultured in the presence (+) or absence (-) of dox (1 g/ml). After 6 days in culture, colonies
were stained for AP activity and scored into three categories (uniformly undifferentiated,
partially differentiated, and fully differentiated) as indicated on the right.
(B) Treatment of ESC nuclear extract with Benzonase removes DNA. One hundred L of
nuclear extract was treated with (+) or without (-) 15 units of Benzonase for 3 hours at 4oC. Size
markers are indicated (MW).
(C) Efficient streptavidin capture (SA-IP) of
bio
Oct4 in ZO4B4 ESCs. Control ZHBTc4 ESCs
cultured in the absence of doxycycline and ZO4B4 ESCs cultured in the presence of doxycycline
were subjected to SA-IP. The precipitated samples were analyzed for expression of the dox
suppressible Oct4 transgene (doxOct4) and biotinylated Oct4 transgene (bioOct4) by western
blotting with anti-Oct4 antibody.
Figure S2. General features of the Oct4 interactome.
(A) Contribution of the Oct4 interactome to a protein interaction sub-network in mESCs. We
constructed an interactome consisting of the Oct4-interacting proteins that were previously
identified as components of the protein complexes of eight pluripotency factors in similar
affinity purification-mass spectrometry (AP-MS) studies. Lists of proteins were extracted from
three published studies3, 5-6. Green nodes are overlapping Oct4 interacting proteins identified by
us and others in the previous Oct4 interactome studies, whereas yellow nodes are proteins newly
identified in this study. The purple nodes are Oct4 interacting proteins from the previous studies
5
only3,
5-6
. The large circles denote the baits for AP-MS studies, and the small circles are the
proteins identified as interacting proteins of these tagged baits.
(B) Co-regulation of Oct4-associated proteins during differentiation of ESCs upon Oct4
depletion by RNAi (top) or during embryoid body (EB) formation (bottom). A selected number
of genes with established roles in self-renewal and pluripotency of ESCs are listed from the
down-regulated cluster. The microarray data for Oct4 depletion and EB differentiation are from
the published studies2, 7.
Figure S3. Enrichment of GO terms and KEGG pathways in the Oct4 interactome.
(A-B) Analysis of the Oct4 interactome for GO terms “molecular function” (A) and “biological
process” (B). Blue and green columns show the percentages of factors in the Oct4 interactome,
respectively.
(C) Analysis of the Oct4 interactome for the “KEGG Pathways”. Black columns show the
numbers of proteins present in the Oct4 interactome.
The p-values for each category are shown on the right. Statistical analysis was carried out using
the mouse genome as the reference dataset.
6
Figure S4. Summary of common protein complexes and factors associated with Oct4 from
three studies.
Comparison of our Oct4 interactome with the two published Oct4 network studies5-6. The details
of the overlapping proteins between our study and the two published studies are listed (see also
Fig. 2B). The common 18 factors among all the three studies are presented in this table with
references provided for their functional studies in ESCs.
NAME
COMPLEX
CHD4
NuRD complex
ESC DEPLETION
PHENOTYPE
-
GATAD2A
NuRD complex
Not detected
Ref8
GATAD2B
NuRD complex
-
MBD3
NuRD complex
MTA2
NuRD complex
Reduced proliferation;
Trophectoderm
differentiation
-
MTA3
NuRD complex
-
-
HDAC1
NuRD complex
Reduced proliferation
Ref13
SMARCA4
SWI/SNF COMPLEX
Differentiation
Ref14
SMARCC1
SWI/SNF COMPLEX
Differentiation
Ref15
KDM1
LSD1 complex
Reduced proliferation
Ref16
RCOR2
LSD1 complex
Reduced proliferation
Ref17
SALL1
Spalt-like transcriptional
repressor complex
Not detected
Ref18
SALL4
Spalt-like transcriptional
repressor complex
Differentiation
Ref19-21
CTBP2
Transcription factor
Increased self-renewal
Ref22-23
HCFC1
Regulation of transcription Differentiation
Ref24
HELLS
Helicases
-
-
OGT
Enzymes
Lethality
Ref25-26
ARID3B
Transcription factor
-
-
7
REFERENCES
-
Ref9-11
Ref12
Figure S5. Enrichment of factors with critical function in stem cell maintenance and early
development in the Oct4 interactome.
Genes encoding the proteins in the Oct4 interactome that have been reported in previous RNAi
knockdown or knockout studies are listed. The available phenotypes are also summarized.
References for the studies are provided. *a “GFP down” indicates no specific phenotype was
described except the observation of downregulation of the Oct4-GFP reporter activity during
RNAi studies in mouse (M) or human (H) ESCs. *b The indicated embryonic days (e.g., E3.5)
are the developmental stage when the knockout embryos die.
SPECIES GENE
NAME
RNAi ESC
PHENOTYPE*a
GFP down
increased selfrenewal
differentiation
KNOCKOUT ESC KNOCKOUT
REFERENCES
PHENOTYPE
EMBRYO
PHENOTYPE*b
viable
Ref27-28
E3.5
Ref29-30
required for
mesoderm
Ref31-32
differentiation
E6.5
~E4
Ref31, Wellcome Trust Sanger Institute, 2010
viable
Ref29, Wellcome Trust Sanger Institute, 2010
Ref27, 29
Ref33, Velocigene
Ref33
Ref31
~E10.5
Ref2, 34-36
Ref29
died after birth
Ref29, 37
Ref24, 29
viable
Ref24, 29, 38
embryonic
Ref29, Wellcome Trust Sanger Institute, 2010
lethality
Ref27, Mammalian Functional Genomics Centre,
E3.5
2010
increased selfRef9, 12, 31, 33-34, 39
renewal
~E8.5
Ref27, 33
M
H
M
Ahnak
Aplp2
Arid1a
GFP down
GFP down
M
H
M,H
M
M
M
M
H
H
H
H
H
Brd4
Cct7
Cnot1
Cpsf1
Cpsf2
Ddx47
Esrrb
Etf1
Hadha
Hcfc1
Helz
Hnrnpu
M
Kif11
M
Mbd3
M
M
Paf1
Rad21
GFP down
GFP down
-
H
M
Rbm17
Rif1
M
Rnf2
differentiation
differentiation
double knockout
with Ring1A
causes
differentiation
no
GFP down
differentiation
differentiation
cell death
cell death
cell death
cell death
differentiation
-
slightly flattened
slight cell death
GFP down
GFP down
GFP down
GFP down
slight cell death
differentiation
GFP down
GFP down
differentiation
GFP down
GFP down
M
H
M
M
M
M
M,H
M,H
M
Sall4
Sf3a3
Smarca4
Smarcc1
Ssrp1
Supt16h
Tcp1
Tpr
Wdr5
33, 40
prenatal lethality Ref
Ref29, Helmholtz Zentrum Muenchen GmbH, 2010
viable
embryonic
Ref31, 33, 35, 41
lethality
Ref27, 31, 33, 42-45
before E10.5
differentiaon
prone
before E 6.5
before E5.5
before E5.5
-
8
Ref19-21, 34
Ref29
Ref14, 31
Ref15, 31
Ref31, 46
Ref31
Ref29, 31
Ref29, 31
Ref27
Supplemental Table Legends
Supplemental Table 1. In Vivo Biotinylation-based Affinity Purification of Oct4 Protein
Complexes in mESCs.
The in vivo biotinylation-based affinity purification of
bio
Oct4-associated protein complexes
followed by mass spectrometry (MS) identification was repeated three times (Exp-I, II, III). The
number of peptides identified for each candidate protein in these independent experiments were
indicated. The numbers in the brackets are the peptides that are also present in BirA control
samples. For example, “7(2)” indicates that there are 7 unique peptides identified in
bio
Oct4
samples and 2 unique peptides in the BirA control samples. When there is no peptide present in
the BirA control samples (which is ideal), only the peptides identified in the biotinylated samples
were indicated. The Oct4 interacting proteins that are shared with the two recently published
studies5-6 and their association with known epigenetic regulatory complexes were also indicated.
Supplemental Table 2. Summary of MS Identification of Oct4-associated Proteins using
Oct4 Antibody-based Affinity Purification.
The native Oct4 antibody-based affinity purification followed by MS identification was
performed in wild-type J1 ESCs. Peptide numbers of immunoprecipitated samples from control
IgG (in the brackets) and anti-Oct4 affinity purifications are indicated. The endogenous Oct4
partners from this study that are also present in our Oct4 interactome are highlighted and
indicated as “Y” (Yes).
9
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