1. Location Analysis of Transcription Factor Binding Tommy Computational Biology Seminar Nov. 2005

2. 2

3. 3 Background Immuno Precipitation ChIP - Chromatin Immuno Precipitation Microarray evolution (from promoter arrays to tiling arrays) ChIP-chip (ChIP followed by microarray hybridization)

4. 4 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)

5. 5 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.

6. 6 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

7. 7 TSS Formation of Pre-Initiation Complex 1.Localization at the promoter 2.DNA melting, initiation and elongation TATABRE IIA TBP IIB TAFs Pol. II IIF IIE IIH Core promoter

8. 8 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 genome NimbleGen 50bp probe every 100bp ABs for Pol II preinitiation complex (PIC) Computational aspects deconvolution of semi-continuous signal

9. 9 Beware, spoiler! The Titanic drowns and Leo DiCaprio dies Kim et al. Map of active promoters

10. 10 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?)

11. 11 Technicalities Follows similar work on ENCODE regions Kim 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 (TAF II 250) of TFIID Kim et al. Map of active promoters * Except for genomic repeats

12. 12 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

13. 13 Kim et al. Map of active promoters

14. 14 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 – H3 Ac, H3K4 Me Kim et al. Map of active promoters

15. 15 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

16. 16 Kim et al. Map of active promoters

17. 17 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

18. 18 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 1 st exon –Some undergo different splicing All at a single cell type! Kim et al. Map of active promoters

19. 19 Transcription machinary vs. Gene Expression 14,437 genes IMR90 human fibroblast cells Compare PIC occupancy to expression Kim et al. Map of active promoters

20. 20 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

21. 21 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

22. 22 Kim, Barrera, Ren et al. Nature (2005) A high-resolution map of active promoters in the human genome So what have we learned?

23. 23 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

24. 24 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

25. 25 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

26. 26 Pancreas β cell

27. 27 Hepatocyte

28. 28 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

29. 29 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

30. 30 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

31. 31 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.

32. 32 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

33. 33 Regulatory network Some differences between regulation in the two tissues Odom et al. HNF regulation in pancreas and liver

34. 34 Regulatory network motifs

35. 35 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

36. 36 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

37. 37 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

38. 38 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

39. 39 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

40. 40 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

41. 41 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

42. 42 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

43. 43 SOX2 NANOG 1271 genes (7%)1687 genes (9%) Boyer et al. Regulation in embryonic stem cells

44. 44 Binding in proximity Co-binding suggests that OCT4, SOX2 & NANOG function together Boyer et al. Regulation in embryonic stem cells

45. 45 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

46. 46 Putative regulatory circuitry Boyer et al. Regulation in embryonic stem cells

47. 47 Boyer et al. Regulation in embryonic stem cells

48. 48 Boyer, Young et al. Cell (2005) Core transcriptional regulatory circuitry in human embryonic stem cells So what have we learned?