June 13, 2008Cited by 705

Authors

First author

• Xi Chen

Corresponding authors

• Chia Lin WEI, Ph.D.

• Group Leader

• Genome Institute of Singapore

• Huck-Hui Ng , Ph.D.

• Executive Director

• National University of Singapore

• 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?

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

How is the ES cell pluripotency maintained?

Main Purpose

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

Main Design

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

Gain insights into the transcriptional regulatory networks in ESC

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

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

Motif Analyses of TFBSs

Matrices predicted by the de novo motif-discovery algorithm Weeder

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

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)

87.4%

56.8%

MTL Associated with Nanog, Oct4, Sox2, Smad1, and STAT3 as ES-Cell Enhanceosomes

The convergence of the two key signaling pathways (via Smad1 and STAT3) with the core circuitry defined by Nanog, Oct4, and Sox2

43.4%

32.9%

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

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

p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster

• 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

the occurrence of p300 in different MTL types

motif from p300-enriched sequences resembles the sox-oct element

ChIP-qPRC of p300

Binding of p300 to the genomic sites depends on Oct4, Sox2 and Nanog

p300 Is Recruited to the Nanog-Oct4-Sox2 Cluster

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

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

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 of Transcription Factors Is Associated with ES-Cell-Specific Expression

Combinatorial binding patterns of TFs have predictive power for ES- cell-specific expression

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

Workflow (1)

• Two tables – Genes ranked by their expression fold change– Genes ranked by their binding score to a TF

Expression-ranked genes list

Binding-ranked genes list

Fold-Change

Big

Small

Gene

TSS

K= 10 kb

Chip-Seq peaks in TSS ± k ?

K <= 1Mb ?

K= K+ 10 kbYes

No

Score = 0

No

Score = # Tags associated with

called peak

Yes

For each TF

TSS

-k +k

Regulatory Network Defining ES-Cell Specific Expression

Expression-ranked genes list

Binding-ranked genes list for a TF

Responder Analysis

Workflow (2)

•

TF Gene

Note: A responder analysis is one in which each subject is classified as either a ‘responder’ or a ‘non-responder’.

Regulatory Network Defining ES-Cell Specific Expression

ESC regulatory network

• 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 coactivator p300 is predominantly recruited to dense binding loci involving proteins found in the first cluster

• Constructed a transcriptional regulartory network model that integrates the two key signaling pathways with the intrinsic factors in ES cells.

Summary

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 alOrganism Mouse Mouse

TF 13 TF+ 2 TR 9 TF

Platform Chip-Seq promoter DNA microarraysAnti-body Target endogenous proteins Target biotin-tagged proteinsNetwork

construction

Chip-Seq + Gene expression Chip-Seq + PPI

Number of networks

1 3

Oct4, Sox2, Klf4,c-Myc,Nanog

Chen et al Kim et al

Dax1, Rex1, Zpf281, Nac1

STAT3, Smad1, Zfx, n-Myc, Esrrb, Tcfcp2l1, E2f1,CTCF

Discussion (2) -- enhancersome

• 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.

Enhanceosome : is a nucleoprotein complex composed of distinct sets of TFs bound directly or indirectly to enhancer DNA

Thanos,D. and Maniatis,T., Cell, 1995Daniel,P. et al, Cell, 2007

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.

Acknowledgements

Pro Ren

Group members:Zehua Liu, Jingyi Wu, Mohamed Nadhir Djekidel

All the audience