The cis regulatory elements

1. Current Topics in Genomics and Epigenomics – Lecture 2 The cis regulatory elements

2. Transcription Process in Eukaryotes

3. Transcription in Eukaryotes • Takes place in the nucleus • The lower strand is used as template • RNA is made from 5’ to 3’ direction • Multiple steps • Class II genes are transcribed by RNAP2 and requires post-transcriptional processing

4. The 5´ Capping • Process: • A GTP is attached to the 5´ end via a 5´-5´ bond (Enzyme:Guanyltransferase) • First few nucleotides (including GTP at N#7) are methylated (Enzyme: Guanine- methyltransferase) • Effects: • Facilitates splicing • Stabilizes the mRNA transcript • Facilitates transport of mRNA to cytoplasm • Recognition by ribosomes in translation

5. Splicing RNA branch point • Splicing and lariat formationvia successive trans-esterification reactions • Two exons are ligated, intron is released as a branched lariat molecule

6. Transcriptional initiation • RNAPII loading to promoters requires general transcription factors (GTF)

7. Transcriptional regulatory elements and their mechanisms of action

8. cis-regulatory elements in the genome Insulator (Enhancer blocking) Insulator (Barrier) Promoter Enhancer Insulator (Barrier) Silencing element

9. Promoters • Promoters are where transcription initiates • Many promoter elements have been found • TATA box was discovered in 1979 by comparison of a number of mRNA start sites

10. How to Locate promoters in the human genome? Cap Analysis of Gene Expression • Determine the full length transcripts and align to the genome

12. crosslink sonicate Chromatin IP input LM-PCR label Hybridize to microarray How to Locate promoters in the human genome? • Determine the full length transcripts and align to the genome • Locate promoters based on binding sites of the pre-initiation complex • ChIP-based analysis is a general approach for mapping transcription factor binding sites

13. Locate promoters in the human genome crosslink sonicate • Locate promoters based on binding sites of the pre-initiation complex • ChIP-chip analysis is a general approach for mapping transcription factor binding sites • ChIP-seq provides a more cost-effective, genome-wide approach Chromatin IP input

14. Locate promoters in the human genome - Kim et al., Nature 2005

15. Sequence features of promoters • CpG island is associated with nearly 80% of the promoters • TATA box is not a general feature of human promoters • Other sequence elements exists

16. cis-regulatory elements in the genome Insulator (Enhancer blocking) Insulator (Barrier) Promoter Enhancer Insulator (Barrier) Silencing element

17. Promoter Enhancers Levine M; Small S.

18. A cis regulatory element of Shh Enhancer Preaxialpolydactyly Lettice et al., Hum Mol Gen 2003, 12 (14) pp. 1725-35 Lettice et al, Proc NatlAcadSci USA 2002 vol. 99 (11) pp. 7548-53

19. RNAP RNAP Model of enhancer function Coactivators

20. p300 is a transcriptional co-activator -- www.web-books.com/ MoBio/Free/Ch4G2.htm

21. Locating enhancers by mapping the p300 binding sites Visel et al., Nature 2009 Z. Li in collaboration with Visel, Pennachio, and Rubin @ LBNL

22. Confirmation of enhancer activities of the p300 binding sites in transgenic mouse embryos • 53/67 (79%) forebrain predictions, 28/34 (82%) midbrain predictions and 20/25 (82%) limb predictions were active in vivo enhancers at e11.5 Visel et al., Nature 2009

23. cis-regulatory elements in the genome Insulator (Enhancer blocking) Insulator (Barrier) Promoter Enhancer Insulator (Barrier) Silencing element

24. Barrier Elements Gaszner and Felsenfeld Nature Reviews Genetics advance online publication; published online 15 August 2006 | doi:10.1038/nrg1925

25. Gaszner and Felsenfeld Nature Reviews Genetics advance online publication; published online 15 August 2006 | doi:10.1038/nrg1925

26. Gaszner and Felsenfeld Nature Reviews Genetics advance online publication; published online 15 August 2006 | doi:10.1038/nrg1925

27. Cohesins and CTCF colocalize on insulators

28. The ENCODE (ENCyclopedia Of DNA Elements) Project • The ENCODE Project was launched in 2003 • Goal: Identify candidate functional elements in the human genome. Produce large scale datasets for public access. • Three phases • Pilot phase (2003-2007) – develop high throughput methods, focusing on 1% of genome • Production phase (2007 – 2012) – ramping up to genome-wide, focusing on a limited number of cell lines; model organisms (worm, fly and mouse) were also included. • Full production phase (2012 – 2016),just launched, aimed for comprehensive analysis. http: //www.genome.gov/ENCODE

29. The ENCODE (ENCyclopedia Of DNA Elements) Project Cis regulatory sequences Genes & Transcripts Myers et al., PLoS Biol. 2011 vol. 9 (4) pp. e1001046

30. ChIP-seq Zhang et al. Genome Biol 2008 crosslink sonicate Chromatin IP input

31. Annotating cis-elements from ChIP-seq data of Transcription factor binding

32. DNase-seq Song & Crawford, Cold Spring HarbProtoc 2010

33. Chromatin State and accessibility in human DNA measured by ChIP-seq and DNase-Seq

35. >80% of the genome participates in one or more biochemical event (making RNA, binding TFs, etc) in at least one cell type; • Defined 399,124 regions with enhancer-like features and 70,292 regions with promoter-like features • 4.5 million Dnase I hypersensitive sites defined • Single nucleotide polymorphisms (SNPs) associated with disease by GWAS are enriched within non-coding functional elements Ecker J, Nature 2012, 489:53

36. Maurano et al… J. A Stamatoyannopoulos, Science 2012