Exploring Genome-wide Organization of Chromatin Structure by ChIP Alon Goren, Ph.D.

Transcription

1 Exploring Genome-wide Organization of Chromatin Structure by ChIP Alon Goren, Ph.D. Broad Institute of MIT & Harvard, MGH Pathology Harvard Medical School

2 Webinar Overview Introduction to chromatin, ChIP (Chromatin IP) and uses of the method Overview of genomic approaches to map in-vivo chromatin structure Detailed description of genome-wide chromatin organization Usage of genome visualization tools Major scientific discoveries stemming from charting of in-vivo chromatin maps Miriam Ferrer, Abcam speaker (highlighting ChIP grade antibodies, ChIP kits) Questions and answers from delegates 2

3 Part I Introduction to chromatin, ChIP (Chromatin IP) and uses of the method Overview of genomic approaches to map in-vivo chromatin structure 3

4 Introduction to Chromatin Structure & Organization Source: Ecker et al., Nature

5 Introduction to Chromatin Structure & Organization Layers of chromatin organization Source: Zhou*, Goren* & Bernstein Nature Reviews Genetics

6 Introduction to ChIP Chromatin ImmunoPrecipitation Goal Map the organization of histone modifications (HMs), transcription factors (TFs) and chromatin regulators (CRs) ChIP 6

7 Overview of ChIP-chip & ChIP-seq ChIP-chip ChIP-seq Source: Schones & Zhao, Nature Reviews Genetics

8 Overview of ChIP-chip & ChIP-seq ChIP-chip ChIP-seq Source: Schones & Zhao, Nature Reviews Genetics

9 Comparison of ChIP-chip and ChIP-seq * * * * * Source: Park Nature Reviews Genetics

10 ChIP-seq a Peak Into the Computational Approach Sequencing Computational pipeline A. Align reads B. Infer positions of ChIP fragments C. Count fragments at each genomic position Source: Mikkelsen et al., Nature

11 Approaches for the Analyses of ChIP-seq Data * * * (WCE) * * * Source: Park Nature Reviews Genetics

12 Part II Detailed description of genome-wide chromatin organization 12

13 Chromatin States Discrete Genomic Combinations of HMs Source: Ernst et al., Nature

14 Chromatin States Discrete Genomic Combinations of HMs TSS Gene body TTS Source: Barth et al., Trends in Bio Sci

15 Five Chromatin Types in Drosophila Cells Source: Filion et al., Cell

16 A Model Fine-Tuning of Genomic Elements by Histone Modifications Source: Zhou*, Goren* & Bernstein Nature Reviews Genetics

17 The Complexity of CRs >100 Isozymes HDACs, HDMs, HMTs, HATs Tissue-specific, developmentally-regulated Amplified, mutated, rearranged in cancer Source: Kouzarides, Cell

18 Part III Usage of genome visualization tools 18

19 Genomic Visualization and Analysis Tools: 3. UCSC: Source: More at 19

20 Viewing ChIP-seq tracks on UCSC Source: 20

21 Genomic Visualization Tools IGV Source: 21

22 Genomic Visualization Tools IGV Source: Jim Robinson, The Broad Institute 22

23 Genomic Visualization Tools IGV Source: Jim Robinson, The Broad Institute 23

24 Genomic Analysis Tools: Genomespace Source: 24

25 Genomespace Principles Source:

26 Genomic Visualization and Analysis Tools: 1. Genomespace Source:

27 Part IV Major scientific discoveries stemming from charting of in-vivo chromatin maps 27

28 Enhancers Identification and Characterization Sources: Heintzman*, Hon* & Hawkins* et al., Nature 2009; Shen*, Yue.* et al., Nature

29 Introduction to Chromatin Regulators (CRs) H3K27me3 H3K4me3 H3K36me3 CRs 29

30 Combinatorial Patterning of Chromatin Regulators (CRs) Source: Ram*, Goren* et al. Cell

31 CRome web portal Source: 31

32 ENCODE Consortium Studies Source: Find more at 32

33 Identification and Characterization Regulatory Regions Source: Neph*, Vierstra*, Stergachis*, Reynolds* et al., Nature

34 Dynamic profiling of TF binding by High-throughput ChIP-seq Source: Garber*, Yosef*, Goren et al., Molecular Cell

35 Future Directions: Developmental Epigenomics 35

36 Acknowledgments Melissa Gymrek Jeff Xing Daniel Fernandez Timothy Durham Oren Ram Noam Shoresh Chuck Epstein Broad Epigenomics Lesley Gafney Dongkeun Jang Janis Lem Alex Meissner Ido Amit Nir Yosef Jason Ernst Jim Robinson Eric Mendenhall Leah Escalante Brad Bernstein Aviv Regev ENCODE consortium Charles H. Hood Foundation Post-doctoral Fellowship Centers of Excellence in Genomic Science (CEGS) 36

37 Part V Miriam Ferrer, Abcam speaker (highlighting ChIP grade antibodies, ChIP kits) 37

38 Protocol and troubleshooting guidebook 2 nd Edition of our general protocol and troubleshooting book ChIP IHC Flow Cytometry and many more Want a copy? Contact: mailrequest@abcam.com Mention Protocol book 38

39 Protocol and troubleshooting guidebook CLIP protocol RIP protocol 39

40 How to contact us Abcam s Scientific Support Team US us.technical@abcam.com Tel: ABCAM (22226) or Website: Hong Kong hk.technical@abcam.com Tel: +(852) Website: UK technical@abcam.com Tel: +44 (0) Website: Japan technical@abcam.co.jp Tel: +81 (0) Website: 40

41 Abcam s new online tool for epigenetics The hottest products The newest protocols The best meetings In one easy location! Bulk up and save Buying in bulk allows you to Save money Minimize variability sales@abcam.com 41

42 EpiSeeker ChIP Kits Your tool for advancing epigenetics 42

43 Available EpiSeeker ChIP kits (All EpiSeeker kits are X-ChIP) General One Step Plant MBD2(Methyl-CpG Binding Domain protein 2) Acetylated histone modifications Optimized for cells or tissue Histone H3 (acetyl) Histone H4 (acetyl) Methylated histone modifications Optimized for cells or tissue Histone H3 (methyl K4) Histone H3 (methyl K9) & (tri-methyl K9) Histone H3 (methyl K27) Methylated DNA MeDIP 5mC (optimized for cells, tissue or nuclear extracts) hmedip 5hmC Resources 43

44 Characteristics EpiSeeker ChIP kits Simple to use Reaction takes place in the wells of the 96-wp: easy for standardization and HTP Quick Only 5 hours to perform ChIP (compared to 2 days using conventional method) Inclusive Kit contains all necessary reagents for ChIP reaction (excluding cross-linking step) Ready to go Pre-selected ChIP-grade antibody: optimized for assay Compatible Eluted DNA can be processed straight away by any DNAamplification method (ChIP-ChIP, ChIP-seq) 44

45 EpiSeeker MeDIP Kits Specific antibody to enrich 5-mC/5- hmc rich DNA regions MeDIP detects 5-mC (methylated cytosine) residues hmedip detects 5-hmC (hydroxymethylated cytosine). 5-hmC is a recently discovered modification in animal cells apparently involved in DNA demethylation, but its role is not clear Ready in less than 4 hours 45

46 Webinar attendee promotion 35% Off ChIP Kits Webinar attendees will receive a discount code for 35% off the following products Ab500 (standard ChIP kit) Any ChIP Kit on the EpiSeeker range 46

47 Chromatin, Replication and Chromosomal Stability 2013 Date: June 17-19, 2013 Location: Copenhagen, Denmark Conference Topics Chromosomal architecture Chromosomal stability Replication structure and fork progression Chromatin maintenance and cellular memory Keynote speakers Susan Gasser and Adrian Bird Share your research Talk and poster places opportunities available Meeting website: 47

48 Questions? 48