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University of Brawijaya 4 th December 2013

University of Brawijaya 4 th December 2013. Austen Ganley INMS. Understanding the Human Genome: Lessons from the ENCODE project. Glossary. Non-coding RNA Sequencing Microarray Transcription start site Active/open Inactive/repression. Genome Genes DNA/RNA Protein Cell

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University of Brawijaya 4 th December 2013

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

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

  3. transcriptional terminator transcriptional start site intron promoter exon

  4. 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

  5. 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

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

  7. Detecting transcription using tiled microarrays

  8. 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)

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

  10. 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

  11. 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

  12. Transcriptional start sites from the DONSON gene Overlapping Genes • 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

  13. 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 www.mun.ca/biochem/courses/3107/Topics/supercoiling.html www.palaeos.com/Eukarya/Eukarya.Origins.5.html

  14. Dnase I hypersensitive sites (DHS) • DNase I preferentially digests nucleosome-depleted regions (DNaseI 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

  15. 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

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

  17. 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

  18. 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 www.nature.com/nrm/index.html

  19. 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

  20. 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

  21. 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

  22. 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

  23. The seven chromatin states

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

  25. ENCODE Grand Summary 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 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

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