June 13, 2008

1. June 13, 2008 Cited by 705

2. Authors First author • Xi Chen Corresponding authors • Huck-Hui Ng , Ph.D. • Executive Director • National University of Singapore • Genome Institute of Singapore • Chia Lin WEI, Ph.D. • Group Leader • Genome Institute of Singapore They focus on stem cell biology and are addressing two questions:1) What makes a stem cell a stem cell?2) How to make a non-stem cell a stem cell?

3. Background Embryonic Stem(ES) Cell • Pluripotency (differentiate into almost all lineages) • Self-renewing ability • Revolutionized biological research through the creation of genetically altered animals. • Human ES cell (promising) • Pluripotency • Self-renewal

4. Main Purpose How is the ES cell pluripotency maintained?

5. Maintenance of Pluripotency • Extrinsic: the LIF and BMP signaling pathways play a central role in the maintenance of a pluripotential stem cell phenotype. • 1. Leukemia Inhibitory Factor (LIF) • The binding of LIF to its receptor activates STAT3 through phosphorylation • 2. Bone Morphogenetic Proteins (BMPs) • The binding of BMP4 to its receptors triggers the phosphorylation of Smad1 and activates the expression of members of the Id(inhibitor of differentiation) gene family • Intrinsic: factors such as transcription factors (TFs) are also essential for specifying the undifferentiated state of ES cells. • Oct4, Sox2, c-Myc, Klf4, Nanog, Esrrb, Zfx

6. Main Design Gain insights into the transcriptional regulatory networks in ESC • Because Transcription factors (TFs) and their specific interaction with targets are crucial for specifying gene expression programs • Objects • 13 transcription factors: Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2I1, E2f1, CTCF • 2 transcription regulators: p300, Suz12 • Methods • ChIP-seq

7. Outline • ES-cell specific enhanceosomes • Mapping of Binding Sites of 13 TFs by Using ChIP-seq • Motif Analyses of TFBSs • A Subset of Multiple Transcription-Factor-Binding Loci • Nanog-Oct4-Sox2 cluster function as ES-Cell enhanceosomes • p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster • ES-cell Regulatory Network • Combinatorial Binding of TFs is correlated with ES-CellSpecific Expression • Regulatory Network Defining ES-Cell Specific Expression

8. Mapping of Binding Sites of 13 TFs by Using ChIP-seq ChIP-seq Determine the threshold through Monte Carlo simulations Remove peaks that were also found in the negative control library Use ChIP-qPCR to further refine the threshold used Valid

9. Motif Analyses of TFBSs Matrices predicted by the de novo motif-discovery algorithm Weeder

10. A Subset of Multiple Transcription-Factor-Binding Loci MTL: Multiple Transcription-Factor-Binding Loci • Plot of the number of TFs bound per co-bound locus. The distribution of randomly occurring co-bound loci is obtained by simulation

11. A Subset of Multiple Transcription-Factor-Binding Loci Distribution of clusters with different numbers of co-bound TFs. (Promoter regions are defined ass sequences 2500 bp upstream and 500 bp downstream of TSS)

12. MTL Associated with Nanog, Oct4, Sox2, Smad1, and STAT3 as ES-Cell Enhanceosomes 32.9% 43.4% 87.4% The convergence of the two key signaling pathways (via Smad1 and STAT3) with the core circuitry defined by Nanog, Oct4, and Sox2 56.8%

13. MTL Associated with Nanog, Oct4, Sox2, Smad1, and STAT3 as ES-Cell Enhanceosomes The binding sites of Nanog group are likely ES-cell specific enhancers

14. MTL Associated with Nanog, Oct4, Sox2, Smad1, and STAT3 as ES-Cell Enhanceosomes The binding of Smad1 and STAT3 depend on Oct4, but otherwise is not

15. p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster the occurrence of p300 in different MTL types • p300 was found to co-occur with the Nanog-Oct4-Sox2 cluster • Most p300-binding sites are associated with 3–6 other TFs • The composition of most p300-containing clusters include Nanog, Oct4, or Sox2

16. p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster ChIP-qPRCof p300 Binding of p300 to the genomic sites depends on Oct4, Sox2 and Nanog motif from p300-enriched sequences resembles the sox-octelement

17. Outline • ES-cell specific enhanceosomes • Mapping of Binding Sites of 13 TFs by Using ChIP-seq • Motif Analyses of TFBSs • A Subset of Multiple Transcription-Factor-Binding Loci • Nanog-Oct4-Sox2 cluster function as ES-Cell enhanceosomes • p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster • ES-cell Regulatory Network • Combinatorial Binding of TFs is correlated with ES-CellSpecific Expression • Regulatory Network Defining ES-Cell Specific Expression

18. Combinatorial Binding of Transcription Factors Is Associated with ES-Cell-Specific Expression Class I genes are enriched in binding sites for Nanog, Oct4, Sox2, Smad1 Class II genes are bound heavily by c-Myc and n-Myc

19. Combinatorial Binding of Transcription Factors Is Associated with ES-Cell-Specific Expression the expression level of classI, class II and III are higher than genes in class IV and class V 60% of genes upregulated in ES cells are from class I and class II Combinatorial binding patterns of TFs have predictive power for ES- cell-specific expression

20. Regulatory Network Defining ES-Cell Specific Expression • Aim: • Construct a network that specifies ES-cells • Dataset: • 2 public undifferentiated v.s differentiated gene expression datasets. • Chip-Seq results generated by this study

21. Workflow (1) Regulatory Network Defining ES-Cell Specific Expression • Two tables • Genes ranked by their expression fold change • Genes ranked by their binding score to a TF Gene TSS K= 10 kb K= K+ 10 kb Yes Chip-Seq peaks in TSS ± k ? Yes Expression-ranked genes list Binding-ranked genes list No Big K <= 1Mb ? For each TF No Fold-Change Score = 0 Score = # Tags associated with called peak Small +k -k TSS

22. Workflow (2) Regulatory Network Defining ES-Cell Specific Expression Expression-ranked genes list Binding-ranked genes list for a TF Note: A responder analysis is one in which each subject is classified as either a ‘responder’ or a ‘non-responder’. Responder Analysis TF Gene

23. ESC regulatory network

24. Summary • TFs are wired to the ES-cell genome in two major ways. • Nanog, Oct4, Sox2, Smad1, STAT3 • c-Myc, n-Myc, Zfx, E2f1 • Highly dense binding loci involving these factors have characteristic features of enhanceosomes • The coactivatorp300 is predominantly recruited to dense binding loci involving proteins found in the first cluster • Constructed a transcriptional regulartorynetwork model that integrates the two key signaling pathways with the intrinsic factors in ES cells.

25. Discussion (1) -- comparison • The authors did a concurrent survey of the location of multiple TF in a single cell type. • Shares some similarities to the work of Kim et al. Chen et al Kim et al STAT3, Smad1, Zfx, n-Myc, Esrrb, Tcfcp2l1, E2f1,CTCF Dax1, Rex1, Zpf281, Nac1 Oct4, Sox2, Klf4,c-Myc, Nanog

26. Discussion (2) -- enhancersome Enhanceosome : is a nucleoprotein complex composed of distinct sets of TFs bound directly or indirectly to enhancer DNA • The study showed that the Nanong-Oct4-Sox2 cluster exhibits features of enhencesome: • The binding sites are densely clustered within relatively compact genomic segments. • These regions act as enhancer when placed downstream of the luciferase reporter. • Associated with active region marks (H3K4me3). • P300 (enhancer marker) is recruited to the Nanog-Oct4-Sox2 cluster. Thanos,D. and Maniatis,T., Cell, 1995 Daniel,P. et al, Cell, 2007

27. Discussion (3) -- weakness • To further confirm the enhancersome's function genome wide and the relationship between the different TFs • - STARR-seq • - biochemical verification • The network might involve more regulatory factors.

28. Acknowledgements Pro Ren Group members: Zehua Liu, Jingyi Wu, Mohamed NadhirDjekidel All the audience