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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 4C 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 4C, 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 Supplemental References 1 Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature genetics 2000; 24:372-376. 2 Ivanova N, Dobrin R, Lu R et al. Dissecting self-renewal in stem cells with RNA interference. Nature 2006; 442:533-538. 3 Wang J, Rao S, Chu J et al. A protein interaction network for pluripotency of embryonic stem cells. Nature 2006; 444:364-368. 4 Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic acids research 1983; 11:14751489. 5 Pardo M, Lang B, Yu L et al. An expanded Oct4 interaction network: implications for stem cell biology, development, and disease. Cell stem cell 2010; 6:382-395. 6 van den Berg DL, Snoek T, Mullin NP et al. An Oct4-centered protein interaction network in embryonic stem cells. Cell stem cell 2010; 6:369-381. 7 Hailesellasse Sene K, Porter CJ, Palidwor G et al. Gene function in early mouse embryonic stem cell differentiation. BMC genomics 2007; 8:85. 8 Marino S, Nusse R. Mutants in the mouse NuRD/Mi2 component P66alpha are embryonic lethal. PloS one 2007; 2:e519. 9 Hendrich B, Guy J, Ramsahoye B, Wilson VA, Bird A. Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development. Genes & development 2001; 15:710-723. 10 Kaji K, Nichols J, Hendrich B. Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells. Development (Cambridge, England) 2007; 134:1123-1132. 11 Zhu D, Fang J, Li Y, Zhang J. Mbd3, a component of NuRD/Mi-2 complex, helps maintain pluripotency of mouse embryonic stem cells by repressing trophectoderm differentiation. PloS one 2009; 4:e7684. 10 12 Liang J, Wan M, Zhang Y et al. Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nature cell biology 2008; 10:731-739. 13 Lagger G, O'Carroll D, Rembold M et al. Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. The EMBO journal 2002; 21:2672-2681. 14 Bultman S, Gebuhr T, Yee D et al. A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Molecular cell 2000; 6:1287-1295. 15 Kim JK, Huh SO, Choi H et al. Srg3, a mouse homolog of yeast SWI3, is essential for early embryogenesis and involved in brain development. Molecular and cellular biology 2001; 21:7787-7795. 16 Wang J, Hevi S, Kurash JK et al. The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation. Nature genetics 2009; 41:125-129. 17 Yang P, Wang Y, Chen J et al. Rcor2 is a Subunit of the LSD1 Complex that Regulates ES Cell Property and Substitutes for Sox2 in Reprogramming Somatic Cells to Pluripotency. Stem cells (Dayton, Ohio) 2011. 18 Nishinakamura R, Matsumoto Y, Nakao K et al. Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development. Development (Cambridge, England) 2001; 128:3105-3115. 19 Elling U, Klasen C, Eisenberger T, Anlag K, Treier M. Murine inner cell mass-derived lineages depend on Sall4 function. Proceedings of the National Academy of Sciences of the United States of America 2006; 103:16319-16324. 20 Yuri S, Fujimura S, Nimura K et al. Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression. Stem cells (Dayton, Ohio) 2009; 27:796-805. 21 Zhang J, Tam WL, Tong GQ et al. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nature cell biology 2006; 8:1114-1123. 11 22 Hildebrand JD, Soriano P. Overlapping and unique roles for C-terminal binding protein 1 (CtBP1) and CtBP2 during mouse development. Molecular and cellular biology 2002; 22:52965307. 23 Tarleton HP, Lemischka IR. Delayed differentiation in embryonic stem cells and mesodermal progenitors in the absence of CtBP2. Mechanisms of development 2010; 127:107-119. 24 Dejosez M, Krumenacker JS, Zitur LJ et al. Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells. Cell 2008; 133:1162-1174. 25 O'Donnell N, Zachara NE, Hart GW, Marth JD. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Molecular and cellular biology 2004; 24:1680-1690. 26 Shafi R, Iyer SP, Ellies LG et al. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proceedings of the National Academy of Sciences of the United States of America 2000; 97:5735-5739. 27 Ding L, Paszkowski-Rogacz M, Nitzsche A et al. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell stem cell 2009; 4:403-415. 28 Lee IH, Lim HJ, Yoon S et al. Ahnak protein activates protein kinase C (PKC) through dissociation of the PKC-protein phosphatase 2A complex. The Journal of biological chemistry 2008; 283:6312-6320. 29 Chia NY, Chan YS, Feng B et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 2010. 30 Rassoulzadegan M, Yang Y, Cuzin F. APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells. The EMBO journal 1998; 17:4647-4656. 31 Fazzio TG, Huff JT, Panning B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 2008; 134:162-174. 12 32 Gao X, Tate P, Hu P, Tjian R, Skarnes WC, Wang Z. ES cell pluripotency and germ-layer formation require the SWI/SNF chromatin remodeling component BAF250a. Proceedings of the National Academy of Sciences of the United States of America 2008; 105:6656-6661. 33 Hu G, Kim J, Xu Q, Leng Y, Orkin SH, Elledge SJ. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes & development 2009; 23:837-848. 34 Kagey MH, Newman JJ, Bilodeau S et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 2010; 467:430-435. 35 Loh YH, Wu Q, Chew JL et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature genetics 2006; 38:431-440. 36 Luo J, Sladek R, Bader JA, Matthyssen A, Rossant J, Giguere V. Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 1997; 388:778-782. 37 Ibdah JA, Paul H, Zhao Y et al. Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death. J Clin Invest 2001; 107:1403-1409. 38 Wagner DS, Gan L, Klein WH. Identification of a differentially expressed RNA helicase by gene trapping. Biochemical and biophysical research communications 1999; 262:677-684. 39 Kaji K, Caballero IM, MacLeod R, Nichols J, Wilson VA, Hendrich B. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nature cell biology 2006; 8:285-292. 40 Tachibana-Konwalski K, Godwin J, van der Weyden L et al. Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes. Genes & development 2010; 24:2505-2516. 41 Buonomo SB, Wu Y, Ferguson D, de Lange T. Mammalian Rif1 contributes to replication stress survival and homology-directed repair. The Journal of cell biology 2009; 187:385-398. 42 Endoh M, Endo TA, Endoh T et al. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity. Development (Cambridge, England) 2008; 135:1513-1524. 13 43 Takihara Y, Tomotsune D, Shirai M et al. Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects. Development (Cambridge, England) 1997; 124:3673-3682. 44 van der Stoop P, Boutsma EA, Hulsman D et al. Ubiquitin E3 ligase Ring1b/Rnf2 of polycomb repressive complex 1 contributes to stable maintenance of mouse embryonic stem cells. PloS one 2008; 3:e2235. 45 Voncken JW, Roelen BA, Roefs M et al. Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition. Proceedings of the National Academy of Sciences of the United States of America 2003; 100:2468-2473. 46 Cao S, Bendall H, Hicks GG et al. The high-mobility-group box protein SSRP1/T160 is essential for cell viability in day 3.5 mouse embryos. Molecular and cellular biology 2003; 23:5301-5307. 14