University of Brawijaya - PowerPoint Presentation

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University of

Brawijaya

4th December 2013

1

Understanding the Human Genome: Lessons from the ENCODE project

Austen Ganley

INMS

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Glossary

Genome

Genes

DNA/RNA

Protein

CellTranscription

Chromatin

Histones

Nucleosomes

Non-coding RNA

Sequencing

Microarray

Transcription start site

Active/open

Inactive/repression

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promoter

t

ranscriptional start site

exon

intron

t

ranscriptional terminator

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Introduction

Individual scientists worked together

Aim was to understand 1% of the human genome (2007), and 100% (2012)

Looked at:

Transcription

Chromatin/transcription factorsReplication

Evolution

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Genes

Now estimated to be about

21,000 protein-coding genes

(taking about 3% of the whole genome)

In addition, there are about 9,000 microRNAs

, and about 10,000 long non-coding RNAs

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Transcription

Transcription was measured by two different methods:

Whole genome microarrays

RNA-sequencing

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Detecting transcription using tiled microarrays

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Transcription

Transcription was measured by two different methods:

Whole genome microarrays

RNA-sequencing

They found at least

62% of the whole genome is transcribed (remember, genes only account for about 3% of the whole genome)

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Transcriptional start sites

Goal is to identify the transcription start sites

N

ot easy to do!

Use a technique called CAGE (

Cap Analysis Gene

E

xpression)

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CAGE

Makes use of the

5’ CAP

on mRNA

First, mRNA is reverse-transcribed, to form cDNA (RNA-DNA hybrid)

Then, biotin is attached to the 5’ CAP, and the cDNA is fragmentedThe biotin fragments are isolated (representing the 5’ end of mRNA), and these fragments are sequenced

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About

60,000

transcription start sites found

Only

half of these match known genes

What do the other ones do? May explain high level of transcriptionThe transcription start sites are often far upstream of the gene start, and can overlap genes

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Overlapping Genes

Transcriptional start sites from

the

DONSON

gene

A

n overlapping gene, starting far upstream

The

DONSON

gene is a known gene

However, some transcripts start in the

ATP50

gene, and include some

ATP50

exons

Two genes are skipped out

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Nucleosomes are formed from DNA that is packaged around histones

Histones are a set of proteins that usually associate as an

octamer

www.palaeos.com/Eukarya/Eukarya.Origins.5.html

www.mun.ca/biochem/courses/3107/Topics/supercoiling.html

Chromatin: histones and nucleosomes

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Dnase

I hypersensitive sites (DHS)

Gilbert,

Developmental Biology

, Sinauer

Hebbes Lab, University of Portsmouth, UK

DNase

I preferentially digests nucleosome-depleted

regions (

DNase

I hypersensitive

sites)

These

are associated with

gene transcription

Chromatin is digested with

DNase

I: only digests nucleosome-free regions

The remaining

DNA is isolated, and put on a

microarray or sequenced

Find the open, active regions of the genome

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DNase

I hypersensitive sites

In total, about

3 million

DNase I hypersensitive sites in the genome, covering about

15% (versus about 40,000 genes covering about 4%)Transcriptional start sites are regions of DNase

I hypersensitivity, as expected

Most

DNase

I hypersensitive sites are not associated with transcriptional start site, though

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Genome

Transcribed region

DNase

I hypersensitive region

Transcription start sites

Genes

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Histone Modification Effects

Modifications occur on the histone tails

They alter the strength of DNA-histone binding, and influence the binding of other proteins to the DNA

Thus they can activate or silence gene expression

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The “Histone Code”

The combination of histone modifications determine

a gene’s transcriptional

status – histone code

Some modifications are associated with active gene

expressionH3K4me2H3K4me3

H3ac

H4ac

Some with repression

H3K27me3

H3K4me1

www.nature.com

/

nrm

/

index.html

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ChIP (Chromatin immunoprecipitation)

Method to find where your protein of interest

is binding to

You cross-link the sample, and fragment the DNA into pieces

Immunoprecipitate

using an antibody to your protein of interest

Reverse the cross-links, and isolate the DNA

To find where in the genome the protein was bound:

Hybridise

the DNA to a

microarray (

ChIP-chip

) OR sequence it (ChIP-seq)

www.rndsystems.com

/

product_detail_objectname_exactachip_assayprinciple.aspx

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Histone modification profiles

They found that histone modifications associated with

active transcription

were found around transcription start sites

They found that histone modifications associated with

gene repression were depleted around transcription start sitesThis is as expected

Around

DNase

I hypersensitive sites not near transcription start sites, they found almost the opposite pattern

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Enrichment of active histone marks and depletion of inactive histone marks at a transcription start site

Enrichment of inactive histone marks but little enrichment of active histone marks at a

DNase

I hypersensitive site

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Histone modification profiles

They also found other patterns

Combining all the results (plus results for transcription factor binding), they say that the human genome is divided into

seven different types of chromatin states

Which state it is depends on what combination of histone modifications/transcription factor binding there is

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The seven chromatin states

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The seven chromatin states

Promoter (red)

Enhancer (yellow)

Gene body (green)

Inactive region (grey)

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Grand Summary

ENCODE

Transcription:

• a lot of non

-coding

transcription

(~60

% of the

genome transcribed) – much more than needed just to transcribe all the genes

Transcription start sites:

•

Twice as many transcription start sites as

traditional “genes

”

• transcripts span large regions, even between

genes

DNase

I hypersensitive sites:

•

more than just at transcription start sites

• two types: those

found both at TSS, and

those found at

other regions

• these have

different chromatin profiles

Histone modifications:

• active marks correlate with TSS/DHS

• distal DHS have a different histone modification profile

Chromatin states:

•

The genome can be divided into seven different types

•

these are determined by the combination of histone modifications and transcription factor binding that occur

Overview:

• genome can be generalised into

seven different states

•

the function of some of these states is known – e.g. promoter

•

the function of others is not known, but may explain the high level of transcription and open chromatin structure