1. Current Topics in Genomics and Epigenomics – Lecture 2

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. RNA branch point Splicing  Splicing and lariat formation via 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)

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

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?  Determine the full length transcripts and align to the genome Cap Analysis of Gene Expression

12. 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  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

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 (Barrier) Promoter Enhancer Insulator (Enhancer blocking) Silencing element Insulator (Barrier)

17. Levine M; Small S. Promoter Enhancers

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

19. Model of enhancer function RNAP 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 Z. Li in collaboration with Visel, Pennachio, and Rubin @ LBNL Visel et al., Nature 2009

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 (Barrier) Promoter Enhancer Insulator (Enhancer blocking) Silencing element Insulator (Barrier)

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

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 Myers et al., PLoS Biol. 2011 vol. 9 (4) pp. e1001046 Genes & TranscriptsCis regulatory sequences

30. ChIP-seq Zhang et al. Genome Biol 2008

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

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

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

35. Ecker J, Nature 2012, 489:53 >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

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