current topics in genomics and epigenomics – lecture 2

The cis regulatory elements

Current Topics in Genomics and Epigenomics – Lecture 2

Transcription Process in Eukaryotes

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

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

RNAbranch point

Splicing

Splicing and lariat formationvia successive trans-esterification reactions

Two exons are ligated, intron is released as a branched lariat molecule

Transcriptional initiation

RNAPII loading to promoters requires general transcription factors (GTF)

Transcriptional regulatory elements and their mechanisms of action

cis-regulatory elements in the genome

Insulator(Barrier)

Promoter Enhancer

Insulator (Enhancer blocking)

Silencing element

Insulator(Barrier)

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

How to Locate promoters in the human genome?

Determine the full length transcripts and align to the genome

Cap Analysis of Gene Expression

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

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

Locate promoters in the human genome

- Kim et al., Nature 2005

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

cis-regulatory elements in the genome

Insulator(Barrier)

Promoter Enhancer

Insulator (Enhancer blocking)

Silencing element

Insulator(Barrier)

Levine M; Small S.

Promoter Enhancers

A cis regulatory element of Shh

Lettice et al., Hum Mol Gen 2003, 12 (14) pp. 1725-35Lettice et al, Proc Natl Acad Sci USA 2002 vol. 99 (11) pp. 7548-53

Enhancer

Preaxial polydactyly

Model of enhancer function

RNAP

RNAP

Coactivators

p300 is a transcriptional co-activator

-- www.web-books.com/ MoBio/Free/Ch4G2.htm

Locating enhancers by mapping the p300 binding sites

Z. Li in collaboration with Visel, Pennachio, and Rubin @ LBNL

Visel et al., Nature 2009

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

cis-regulatory elements in the genome

Insulator(Barrier)

Promoter Enhancer

Insulator (Enhancer blocking)

Silencing element

Insulator(Barrier)

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

Barrier Elements

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

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

Cohesins and CTCF colocalize on insulators

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

The ENCODE (ENCyclopedia Of DNA Elements) Project

Myers et al., PLoS Biol. 2011 vol. 9 (4) pp. e1001046

Genes & TranscriptsCis regulatory sequences

ChIP-seq Zhang et al. Genome Biol 2008

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

DNase-seq

Song & Crawford, Cold Spring Harb Protoc 2010

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

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

Maurano et al… J. A Stamatoyannopoulos, Science 2012