Genomics of Gene Regulation

1. Genomics of Gene Regulation ANSC 497B Ross Hardison Nov. 10, 2009

2. DNA sequences involved in regulation of gene transcription Protein-DNA interactions Chromatin effects

3. Distinct classes of regulatory regions Act in cis, affecting expression of a gene on the same chromosome. Cis-regulatory modules (CRMs) Maston G, Evans S and Green M (2006) Annu Rev Genomics Hum Genetics 7:29-59

4. RNA polymerase II TATA box + Initiator: Core or minimal promoter. Site of assembly of preinitiation complex Upstream regulatory elements: Regulate efficiency of utilization of minimal promoter Maston, Evans & Green (2006) Ann Rev Genomics & Human Genetics, 7:29-59 General features of promoters • A promoter is the DNA sequence required for correct initiation of transcription • It affects the amount of product from a gene, but does not affect the structure of the product. • Most promoters are at the 5’ end of the gene.

5. Conventional view of eukaryotic gene promoters Maston, Evans & Green (2006) Ann Rev Genomics & Human Genetics, 7:29-59

6. CpG island, no TATA About 90% of promoters Carninci … Hayashizaki (2006) Nature Genetics 38:626 Most promoters in mammals are CpG islands TATA, no CpG island About 10% of promoters

7. Differences in specificity of start sites for transcription for TATA vs CpG island promoters Fraction of mRNAs Carninci … Hayashizaki (2006) Nature Genetics 38:626

8. CRM pr luciferase UCE pr lacZ Tested UCE Over half of ultraconserved noncoding sequences are developmental enhancers Pennacchio et al. (2006) Nature 444:499-502 Pennacchio et al., http://enhancer.lbl.gov/ About half of the enhancers predicted by interspecies alignments are validated in erythroid cells Wang et al. (2006) Genome Research 16:1480- 1492 Enhancers • Cis-acting sequences that cause an increase in expression of a gene • Act independently of position and orientation with respect to the gene.

9. CRMs are clusters of specific binding sites for transcription factors Hardison (2002) on-line textbook Working with Molecular Genetics http://www.bx.psu.edu/~ross/

10. Ex1 Ex2 Enhancers can occur in a variety of positions with respect to genes Enhancer Enhancer P Transcription unit Upstream Adjacent Downstream Internal Distal

11. Silencer • Cis-acting sequences that cause a decrease in gene expression • Similar to enhancer but has an opposite effect on gene expression • Gene repression - inactive chromatin structure (heterochromatin) • SIR proteins (Silent Information Regulators) • Nucleates assembly of multi-protein complex • hypoacetylated N-terminal tails of histones H3 and H4 • methylated N-terminal tail of H3 (Lys 9)

12. Neo-resistant colonies % of maximum Insu- lator Pr neoR Enhancer 10 50 100 Silencer Insulators and boundaries • A boundary in chromatin marks a transition from open to closed chromatin • An insulator blocks activation of promoter by an enhancer • Requires CTCF • Example: HS4 from chick HBB complex has both functions

13. me3 K27 H3 N-tail Repression by PcG proteins via chromatin modification Polycomb Group (PcG) Repressor Complex 2: ESC, E(Z), NURF-55, and PcG repressor SU(Z)12 Methylates K27 of Histone H3 via the SET domain of E(Z) OFF

14. trx group (trxG) proteins activate via chromatin changes • SWI/SNF nucleosome remodeling • Histone H3 and H4 acetylation • Methylation of K4 in histone H3 • Trx in Drosophila, MLL in humans • http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html#Part_3 Me1,2,3 K4 ON H3 N-tail

15. Histone modifications modulate chromatin structure H3K4me2, 3 H3K27me3 Uta-Maria Bauer http://www.imt.uni-marburg.de/bauer/images/fig2.jpg

16. Repressed and active chromatin Dustin Schones and Keiji Zhao (2008) Nature Reviews Genetics 9: 179

17. Pol IIa Biochemical features of DNA in CRMs Accessible to cleavage: DNase hypersensitive site Clusters of binding site motifs Bound by specific transcription factors Coactivators Pol II Nucleosomes with histone modifications: Acetylation of H3 and H4 Methylation of H3K4 Lack of methylation at H3K27 or H3K9 … Associated with RNA polymerase and general transcription factors

18. Methods in Genomics of Gene Regulation

19. Chromatin immunoprecipitation: Greatly enrich for DNA occupied by a protein Elaine Mardis (2007) Nature Methods 4: 613-614

20. ChIP-chip: High throughput mapping of DNA sequences occupied by protein http://www.chiponchip.org Bing Ren’s lab

21. Enrichment of sequence tags reveals function Barbara Wold & Richard M Myers (2008) “Sequence Census Methods” Nature Methods 5:19-21

22. Illumina (Solexa) short read sequencing - 8 lanes per run - 10 M to 20 M reads of 36 nucleotides (or longer) per run. - 1 lane can produce enough reads to map locations of a transcription factor in a mammalian genome.

23. Example of ChIP-seq ChIP vs NRSF = neuron-restrictive silencing factor Jurkat human lymphoblast line NPAS4 encodes neuronal PAS domain protein 4 Johnson DS, Mortazavi A, Myers RM, Wold B. (2007) Genome-Wide Mapping of in Vivo Protein-DNA Interactions. Science316:1497-1502.

24. ChIP-seq for chromatin modifications Dustin Schones and Keiji Zhao (2008) Nature Reviews Genetics 9: 179

25. Histone modifications around HBB locus Known CRMs UCSC genes trithorax Polycomb Transcription associated mark DNase hyper- sensitive sites

26. Distributions at all GenCode TSSs Symmetrical distribution of: - H3K4me3, H3K4me2 - H3Ac, H4Ac, DHS - E2F1, E2F4, Myc, Pol II Birney et al. (2007) Nature477: 799-816

27. Distribution of histone modifications and factor binding around regulatory regions • Promoters • H3K4me3, H3K4me2 • E2F1, E2F4, Myc, Pol II • Distal HSs • H3K4me1: enhancers • CTCF: insulators Birney et al. (2007) Nature, 447:799-816

28. Enhancers predicted from chromatin signatures (2009) Nature 459: 108-112

29. Enhancer predictions in human cells

30. Characteristics and validation of predicted enhancers

31. Data Resources for Genomics of Gene Regulation

32. UCSC Genome Browser • Visualize data described in publications, e.g. • Expression data • Affymetrix gene arrays, GNF, Su et al. 2004 • Regulation • Kim et al. 2005, PICs (TAF1) • Kim et al., 2008, CTCF • Boyle et al., 2008, DNase hypersensitive sites • Heintzman et al., 2009, Enhancers predicted by H3K4me1 • Mikkelsen et al., 2007, Chromatin modifications in pluripotent and lineage-committed cells • ENCODE project, Production phase • Expression • Affy high density tiling arrays • RNA-seq from several sources (CSHL, Helicos) • Regulation • Broad histone modifications • HAIB DNA methylation • Open Chromatin • UW DNase HS • HAIB TFBS • Yale TFBS • SUNY RBP

33. Locus control region HS5 4 3 2 1 Factor occupancy and DNase hypersen-sitivity ENCODE Tracks: Broad histone modifications, Open chromatin, UW DHS, Yale TFBSs

34. Collated sets of published regulatory regions • http://www.bx.psu.edu/~ross/dataset/Reguldata.html • Noncoding DNA segments with high regulatory potential • PRPs: Intersection of the High RP segments and the PReMods (clusters of conserved transcription factor binding site motifs) • Most constrained DNA segments, phastCons • DNase hypersensitive sites in CD4+ T cells • DNA segments occupied by CTCF in primary fibroblasts • Preinitiation complexes (TAF1) in IMR90 cells • Predicted erythroid cis-regulatory modules

35. GeneTrack • Genomic data analysis and integration • Istvan Albert, Frank Pugh, et al., PSU • http://genetrack.bx.psu.edu/ • Install on your system • Gallery of data for visualization • Yeast H2AZ nucleosome predictions, 454 sequencing • Drosophila H2AZ nucleosome predictions, 454 sequencing

36. Yeast nucleosome map

37. HIS3: nucleosome-free region

38. modENCODE http://www.modencode.org/ Worm and Fly Gene annotations Expression Chromatin modifications TFBs in vivo, etc.

39. Experimental Tests in the Genomics of Gene Regulation

40. G1E-ER4 cells GATA-1 is required for erythroid maturation Common myeloid progenitor Hematopoietic stem cell MEP G1E cells GATA-1 Common lymphoid progenitor Myeloblast Eosinophil Basophil Monocyte, macrophage Neutrophil Aria Rad, 2007 http://commons.wikimedia.org/wiki/Image:Hematopoiesis_(human)_diagram.png

41. GATA1-induced changes in gene expression and occupancy genome-wide Occupancy by TFs and histone modifications along a 60 Mb region Genes induced or repressed after restoration of GATA1

42. High sensitivity and specificity of high throughput occupancy data

43. High throughput occupancy matches known CRMs at Hbb locus

44. Confirmed and novel regulatory regions for Gypa Known CRMs Gypa gene Response DHSs GATA1 TAL1 Trx: H3K4me1 Trx: H3K4me3 PcG: H3K27me3 Input DNA

45. Induced genes have GATA1 occupied segments close to their TSS

46. DNA segments occupied by GATA-1 were tested for enhancer activity on transfected plasmids Occupied segments

47. Some of the DNA segments occupied by GATA-1 are active as enhancers Cheng et al. (2008) Genome Research 18:1896-1905

48. Binding site motifs in occupied DNA segments can be deeply preserved during evolution Consensus binding site motif for GATA-1: WGATAR or YTATCW 5997 constrained 7308 not constrained 2055 no motif

49. All GATA1-occupied segments active as enhancers are also occupied by SCL and LDB1

50. Genetic Determinants of Variation in Gene Expression