location analysis of transcription factor binding

Location Analysis of Transcription Factor Binding

TommyComputational 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)

TFIIA2-3 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 promoter2. DNA melting, initiation and elongation

TATA BRE

IIA TBP IIBTAFs

Pol. IIIIF

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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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


* 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)


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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


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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


possible genes

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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)


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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!


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


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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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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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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


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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


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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).


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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


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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%)


– 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


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Regulatory network

• Some differences between regulation in the two tissues


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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]


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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


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Regular Chain motifs

• Regulator chain motifs represent the simplest circuit logic for ordering transcriptional events in a temporal sequence


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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


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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


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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


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SOX2 NANOG1271 genes (7%) 1687 genes (9%)


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Binding in proximity

• Co-binding suggests that OCT4, SOX2 & NANOG function together


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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


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Putative regulatory circuitryBoyer 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?

location analysis of transcription factor binding

Documents

map of active promoterskim

putative promoters

bound promotersbound

genes dbtss

human genomefound

arrayschipchip chip

bound promotersstandard

chip design