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