Location Analysis of Transcription Factor Binding Tommy Computational Biology Seminar Nov. 2005.
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Transcript of Location Analysis of Transcription Factor Binding Tommy Computational Biology Seminar Nov. 2005.
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Location Analysis of Transcription Factor Binding
Tommy
Computational Biology Seminar
Nov. 2005
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Background• Immuno Precipitation• ChIP - Chromatin Immuno Precipitation• Microarray evolution
(from promoter arrays to tiling arrays)
• ChIP-chip (ChIP followed by microarray hybridization)
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Things to do with ChIP chip…
General method for identification of
– Target genes of transcription factors– Transcribed genes (Pol II)
– Transcribed miRNAs (Pol II)
– Chromatin states (ABs for modified histones)
– etc. – (any protein (mod AB) that binds DNA)
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Outline
• Kim, Ren et al. Nature (2005)A high-resolution map of active promoters in the human genome.
• Boyer, Young et al. Cell (2005)Core transcriptional regulatory circuitry in human embryonic stem cells.
• Odom, Young et al. Science (2004)Control of pancreas and liver gene expression by HNF transcription factors.
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General Transcription Factors (GTFs)
TFIIA
2-3 subunits
TFIIE
2 subunits
TFIIB
1 subunit
TFIID
15 subunits
TFIIF 2 subunits
Pol II
12 subunits
TFIIH
9 subunits
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TSS
Formation of Pre-Initiation Complex
1. Localization at the promoter
2. DNA melting, initiation and elongation
TATA BRE
IIATBP IIB
TAFsPol. II
IIF
IIE IIH
Core promoter
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Kim, Barrera, Ren et al. Nature (2005)A high-resolution map of active promoters
in the human genome
• Accurate mapping of active promoters in human fibroblast cells (IMR90)– Active genes– Identify transcription start sites
• DNA microarray of Human genomeNimbleGen 50bp probe every 100bp
• ABs for Pol II preinitiation complex (PIC)• Computational aspects
deconvolution of semi-continuous signal
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Beware, spoiler!
The Titanic drowns and Leo DiCaprio dies
Kim et al. Map of active promoters
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Kim, Barrera, Ren et al. Nature (2005)A high-resolution map of active promoters
in the human genome
• Found 12,150 bound regions (promoters)– 10,576 belong to 6,763 known genes– 1,196 un-annotated transcriptional units
• Many genes with multiple promoters
• Clusters of active promoters
• Four classes of promoters
• Many novel genes (RNA genes?)
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Technicalities
• Follows similar work on ENCODE regionsKim et al, Gen. Res. (2005); ENCODE project, Science (2004)
• Chip design: series of DNA microarrays covering 14.5 million (!) 50bp probes, covering all the human genome*
• IP design: Monoclonal AB to TAF1 (TAFII250) of TFIID
Kim et al. Map of active promoters
* Except for genomic repeats
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Method
• Compare IP to control DNA• Identify stretches of 4 bound probes• Re-check using a new array• Computational detection of 12,150
peaks (Mpeak)• Compare to known genes
(DBTSS, RefSeq, GenBank, EnsEMBL)
• 87% matched 5’ ends of known mRNAs (up to 2.5Kb)
Kim et al. Map of active promoters
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Kim et al. Map of active promoters
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Validation of results
• Anti-RNAP AB re-found 97% of bound promoters
• Standard ChIP found 27/28 of randomly selected bound promoters
• Bound promoters are enrichment for known TSS elements
• 97% of promoters had chromatin state of active genes – H3Ac, H3K4Me
Kim et al. Map of active promoters
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Un-annotated promoters
• 1,597 promoters are ≥ 2.5Kb from 5’ of known genes
• 607 of them match EST
• 632 of them are also bound by RNAP and in the “right” chromatin state– Measure mRNA expression of 567 promoters
(50bp probes at 28Kb around each gene) – 35 new transcription units. Rest unstable?– One located 250bp ups to predicted miRNA
Kim et al. Map of active promoters
possible genes
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Kim et al. Map of active promoters
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Un-annotated promoters
• 1,239 putative promoters correspond to novel transcription units.– Evolutionary conserved– Enriched with core promoter motifs
• 1,196 outside current gene annotation(13% of promoters)
Kim et al. Map of active promoters
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Clusters of active genes
• 256 clusters of ≥4 active genes(1,668 EnsEMBL genes)
• 1609 genes had multiple promoters– Most have the same gene product– Some have different 1st exon– Some undergo different splicing
• All at a single cell type!
Kim et al. Map of active promoters
1
10
100
1000
10000
2 3 4 5
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Transcription machinaryvs.
Gene Expression• 14,437 genes
• IMR90 human fibroblast cells
• Compare PIC occupancy to expression
Kim et al. Map of active promoters
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• Classes I and IV are consistent (75% of genes)
• Class II - PIC is bound, no expression– PIC is assembled but not sufficient for TXN
• Contain immediate response genes (stress)
– mRNA transcribes but degraded (miRNA targets?)
• Class III - Expressed with no bound PIC– Test 10 random genes with ChIP (TFIID, RNAP)
– Nearly 60% were weakly bound
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Kim, Barrera, Ren et al. Nature (2005)A high-resolution map of active promoters
in the human genome
• Found 12,150 bound regions (promoters)
• Many genes with multiple promoters
• 1,239 novel genes (RNA genes?)
• Clusters of active promoters (chromatin)
• Four classes of promoters
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Kim, Barrera, Ren et al. Nature (2005)A high-resolution map of active promoters
in the human genome
• So what have we learned?
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Odom, Young et al. Science (2004)Control of pancreas and liver gene
expression by HNF transcription factors
• Diabetes is bad.
• Uncover the transcriptional regulatory network that control insulin secretion.
• Human liver and pancreatic islets
• Use ChIP for Pol II and 3 TFs
• Measure expression of genes
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Background
• Transcriptional regulation in the liver– HNF1α (homeodomain)– HNF4α (nuclear receptor)– HNF6 (onecut)
• Same with the pancreatic islets?– All three are require for normal function– Mutations maturity-onset diabetes of the
young (MODY3, MODY1)
• Understand normal to explain abnormal
Odom et al. HNF regulation in pancreas and liver
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MODY• maturity-onset diabetes of the young
• Genetic disorder of the insulin-secreting pancreatic β cells
• Onset of diabetes mellitus before 25
• Autosomal dominant pattern of inheritance
• Not to confuse with type 2 (late-onset) diabetes– early-onset insulin resistance– functional defects in insulin secretion
Odom et al. HNF regulation in pancreas and liver
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Pancreas β cell
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Hepatocyte
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Method
• Identify targets of three TFs in two tissues
• Identify transcribed genes (using Pol II)
• Promoter array (13K genes)
• -700bp to +200bp relatively to TSS
Odom et al. HNF regulation in pancreas and liver
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Hepatocyte targets of HNF1α
• 222 genes that represent a substantial section of hepatocyte biochemistry– gluconeogenesis and associated pathways– carbohydrate synthesis and storage– Lipid metabolism
(synthesis of cholesterol and apolipoproteins)
– Detoxification(synthesis of cytochrome P450 monooxygenases)
– Serum proteins(synthesis of albumin and coagulation factors).
Odom et al. HNF regulation in pancreas and liver
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Pancreas targets of HNF1α
• 106 genes, 30% of which bound in liver• Fewer chaperons and enzymes• Receptors and signal transduction genes vary• Many known targets are missing…
– Stringent criteria– Short promoters
Odom et al. HNF regulation in pancreas and liver
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Targets• HNF6 binds 227 (1.3%) and 189 (1.45%),
incl. important cell-cycle regulators
• HNF4α 1575 (12%) and 1423 (11%)
Odom et al. HNF regulation in pancreas and liver
– Two different ABs– Western blots– Standard ChIP (50)
– Other tissues (17)
– Preimmune ABs bind not– 80% (73%) also bound by PolII.
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The transcriptome• “It is difficult to determine the transcriptome of these
tissues accurately by profiling transcript levels with DNA microarrays.”
• What is the appropriate reference RNA?
• 2,984 (23%) are bound by Pol II in hepatocytes
• 2,426 (19%) in islets, 81% of which by both
• 80% (73%) of HNF4α are bound by Pol II
• Three HNFs cover many of transcribed genes
Odom et al. HNF regulation in pancreas and liver
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Regulatory network
• Some differences between regulation in the two tissues
Odom et al. HNF regulation in pancreas and liver
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Regulatory network motifs
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Multi-component loop
• Capacity for feedback control and produce bistable systems that can switch between two alternate states [Milo et al, 2002]
• The multi-component loop of HNF1α and HNF4α is responsible for stabilization of the terminal phenotype in pancreatic beta cells [Ferrer 2002]
Odom et al. HNF regulation in pancreas and liver
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Feed-forward loop
• A feedforward loop acts as a switch, sensitive to sustained inputs (rather than transient)
• HNF6 serves as a master regulator for feed-forward motifs in hepatocytes and pancreatic islets
• Involves >80 genes in each tissue
Odom et al. HNF regulation in pancreas and liver
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Regular Chain motifs
• Regulator chain motifs represent the simplest circuit logic for ordering transcriptional events in a temporal sequence
Odom et al. HNF regulation in pancreas and liver
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Summary
• HNF4α binds almost half of active genes in the liver and pancreas islets
• Crucial for development and function of these tissues
• Might explain why mutations can increase type II diabetes
Odom et al. HNF regulation in pancreas and liver
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Boyer, Young et al. Cell (2005)Core transcriptional regulatory circuitry
in human embryonic stem cells
• Embryonic stem cells are important– Can be propagated in undifferentiated state – Can differentiate into >200 unique cell types– Great promise for regenerative medicine
• Reveal transcriptional regulatory circuitry controlling pluripotency and self-renewal.
• Early development and cell identity is controlled by several homeodomain TFs
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Background• Early development and cell identity is
controlled by several homeodomain TFs
• OCT4, SOX2, NANOG have central roles in maintaining the pluripotency of stem cells
• KO of each results with differentiation
• Over-expression of OCT4 ~ NANOG KO
• Why? Identify targets of each and see…
Boyer et al. Regulation in embryonic stem cells
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Method• Human H9 embryonic stem cells
• Agilent promoter arrays– 60-mer probes– Spaced at ~300bp– Covering -8Kb to +2Kb relatively to TSS
• Including 98% of TRANSFAC binding sites (Wow!!)
– 17,917 genes
• Replicate set of ChIP assay
Boyer et al. Regulation in embryonic stem cells
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OCT4
Analysis of peaks found:• 623 genes (3%)• 5 miRNAs (3%)
Many known targets:• Mouse ES cells• Expressed in ES
Improved protocol• Better than Odom et al• <1% FPR, 20% FNR
Boyer et al. Regulation in embryonic stem cells
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SOX2 NANOG
1271 genes (7%) 1687 genes (9%)
Boyer et al. Regulation in embryonic stem cells
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Binding in proximity
• Co-binding suggests that OCT4, SOX2 & NANOG function together
Boyer et al. Regulation in embryonic stem cells
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Function of TFs
• Checked expression these genes in ES cells (published data)
• 1,303/2,260 genes are active, 957 inactive
• Of the 353 tri-bound genes, half active
• Active include TFs (OCT4, SOX2, NANOG, STAT3, ZIC3), components of TGF-β and Wnt pathways
• Inactive genes include developmental TFs (important for differentiation)
• Many other homeodomain TFs
Boyer et al. Regulation in embryonic stem cells
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Putative regulatory circuitry
Boyer et al. Regulation in embryonic stem cells
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Boyer et al. Regulation in embryonic stem cells
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Boyer, Young et al. Cell (2005)Core transcriptional regulatory circuitry
in human embryonic stem cells
• So what have we learned?