1. Understanding the Human Genome: Lessons from the ENCODE project
University of Brawijaya
4th December 2013
Austen Ganley
INMS
Understanding the Human Genome: Lessons from the ENCODE project

3. Glossary Genome Non-coding RNA Genes Sequencing DNA/RNA Microarray
Protein
Cell
Transcription
Chromatin
Histones
Nucleosomes
Non-coding RNA
Sequencing
Microarray
Transcription start site
Active/open
Inactive/repression

4. transcriptional start site transcriptional terminator
intron
promoter
exon

5. Introduction Individual scientists worked together
Aim was to understand 1% of the human genome (2007), and 100% (2012)
Looked at:
Transcription
Chromatin/transcription factors
Replication
Evolution

6. 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

7. Transcription Transcription was measured by two different methods:
Whole genome microarrays
RNA-sequencing

8. Detecting transcription using tiled microarrays

9. 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)

10. Transcriptional start sites
Goal is to identify the transcription start sites
Not easy to do!
Use a technique called CAGE (Cap Analysis Gene Expression)

11. 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 fragmented
The biotin fragments are isolated (representing the 5’ end of mRNA), and these fragments are sequenced

12. 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 transcription
The transcription start sites are often far upstream of the gene start, and can overlap genes

13. Overlapping Genes An overlapping gene, starting far upstream
Transcriptional start sites from the DONSON gene
An 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

14. Chromatin: histones and nucleosomes
Nucleosomes are formed from DNA that is packaged around histones
Histones are a set of proteins that usually associate as an octamer

15. Dnase I hypersensitive sites (DHS)
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
Hebbes Lab, University of Portsmouth, UK
Gilbert, Developmental Biology, Sinauer

16. 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

17. Transcription start sites
Genome
Transcribed region
Transcription start sites
DNase I hypersensitive region
Genes

18. 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

19. The “Histone Code”
The combination of histone modifications determine a gene’s transcriptional status – histone code
Some modifications are associated with active gene expression
H3K4me2
H3K4me3
H3ac
H4ac
Some with repression
H3K27me3
H3K4me1

20. 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)

21. 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 sites
This is as expected
Around DNase I hypersensitive sites not near transcription start sites, they found almost the opposite pattern

22. 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

23. 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

24. The seven chromatin states

25. The seven chromatin states
Enhancer (yellow)
Gene body (green)
Inactive region (grey)
Promoter (red)

26. Grand Summary ENCODE 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
Transcription:
• a lot of non-coding transcription (~60% of the genome transcribed) – much more than needed just to transcribe all the genes
ENCODE
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
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