A Biotechniques Webinar Seminar on Epigenetics and the Histone Code

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Brian D. Strahl's lecture presented in the BIOTECHNIQUES VIRTUAL SYMPOSIUM on "The Cell Landscape: From Genotype to Phenotype" Wednesday, October 3, 2012

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A Biotechniques Webinar Seminar on Epigenetics and the Histone Code

  1. 1. BioTechniques Symposium: The Genome and BeyondUnderstanding the role of epigenetic change in gene regulation October 5th, 2011 Histone modifications and their influence on effector protein and antibody recognition Brian D. Strahl Department of Biochemistry & Biophysics UNC-School of Medicine Chapel Hill, NC 27599
  2. 2. Transcriptional regulation Set2 Rad6 Set1 (H3-K36) (H2B-ub) (H3-K4)
  3. 3. Organization of eukaryotic chromatin DNA double helix Histone NucleosomesH3H2B SolenoidH2AH4 Chromatin loop: ~100,000 bp DNA Chromatin
  4. 4. Molecular mechanisms that affect chromatin structure 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications Acetylation Phosphorylation Methylation ADP-ribosylation Ubiquitylation Sumoylation 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation
  5. 5. Molecular mechanisms that affect chromatin structure 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications Acetylation Phosphorylation Lysines (mono-, di-, tri-methyl) Methylation Arginines (mono, di-) ADP-ribosylation Ubiquitylation Sumoylation 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation
  6. 6. Histone ModificationsAcetylationPhosphorylationMethylation Histone Code Chromatin regulatorADP-ribosylationUbiquitinationSumoylation Tail Globular
  7. 7. Epigenetic ‘Toolkit’Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful languagewhere context matters. J. Mol. Biol. 409:36-46.
  8. 8. Histone ModificationsAcetylationPhosphorylationMethylation Histone Code Chromatin regulatorADP-ribosylationUbiquitinationSumoylation Tail Globular
  9. 9. Bromodomain-containing proteins can bind to acetylated histones! (TBP) TATAA (adapted from E. Pennisi - Science, 2000)
  10. 10. Histone Code readers DDT PHD PHD bromo BPTF (NURF)PWWP Jmjc PHD PHD tudor tudor JMJD2A Jmjn (demethylase) bromo bromo bromo bromo bromo bromo BAH BAH HMG BAF180 (SWI/SNF) PHD PHD chromo chromo DEXD HELIC CHD4 (NuRD) (Figure from Abcam)
  11. 11. Mechanisms of histone recognitionGardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful languagewhere context matters. J. Mol. Biol. 409:36-46.
  12. 12. Peptide microarrays
  13. 13. Generation of a combinatorially-modified histone Histone Peptides library peptide me2s me3 me3 me2a me2 me2 me1 me1 me1 me3 cit ac ac ac ac A R T K Q T A R K S T G G K A P R K Q L - K(biotin)-NH2 H3(1-20) phos phos phos me3 me2 me1 ac K S A P S T G G V K K P H R Y K P G T - Peg - K(biotin)-NH2 H3(27-45) me3 me3 A P R K Q L A T K A A R K S A P S T G G V K K P H R Y - G G - K(biotin)-NH2 H3(15-41) me3 me2 me1 acac-I A Q D F K T D L R F - Peg - K(biotin)-NH2 H3(74-84) phos ac ac ac acac-S G R G K Q G G K A R A K A K T R - Peg - K(biotin) H2A(1-17) me2a ac ac ac ac ac P E P A K S A P A P K K G S K K A V T K A Q K K - Peg - K(biotin) H2B(1-24) ac ac ac phos ac ac ac ac me2ac-S G R G K G G K G L G K G G A K R H R K V L R - Peg - K(biotin) H4(1-23)
  14. 14. Design of our histone peptide arrays Thanks to… Or Gozani
  15. 15. Are histone modification-specific antibodies really, um, specific?
  16. 16. Methyl-specific antibodies tend to recognize multiple methylation states H3K4me H3K9meH3K18me H3K36me H3K79me Peptides - 1 2 3 1 2 3 1 2 3 - 1 2 3 - 1 2 3 !K4me1 1.0 Antibodies !K4me2 0.8 0.6 !K4me3 0.4 0.2 !K79me1 0 scale !K79me2 !K79me3 1.2 H3K4me0 *** H3K4me1 *** 1.0 H3K4me2 normalized intensity H3K4me3 0.8 0.6 0.4 0.2 * 0.0 me0 me1 me2 me3 me0 me1 me2 me3 me0 me1 me2 me3 ! H3K4me1 ! H3K4me2 ! H3K4me3
  17. 17. Histone Abs can have cross-target affects – c ac /1 ac K1 ac 6ac 6a /18 5 K5 3/1 6 /1 K3 /14 K1 2 /1 K1 c K4 ac K8 ac 4a K9 c K1 c K5 c – ac /9 /9 /8 a a 8 2 a K4 K5 – abcam !H3K14ac millipore active motif !H4tetra acetyl H3 H2A H3K14 H4 STGG-KAPRK H3(10-18) |||| | | ! STGGVKKPHR H3(31-40) H3K36
  18. 18. Are histone antibodies influenced by neighboring PTMs?
  19. 19. Neighboring modifications influence Histone antibody recognition K9 3 K9 2 e1 e e ac m m m K9 K9 ac 1.0 – K9 H3S10phos – 0.8!H3S10phos 0.6 0.4 !H3K9ac 0.2 S10phos 0 scale
  20. 20. Neighboring modifications can alter H3K4me3 Interactio Neighboring modifications alter antibody recognition H3K4me3 recognition speci H3K4 me0 me3 me0 me3 me0 me3 H3 (1-20) R2me1 R2me1 K9ac K14ac K18ac R2me2s 1.0 R2me2a 0.8 R2me2a K9ac K14ac K18ac 0.6 R2me2a S10phos R2me2a S10phos K9ac K14ac K18ac 0.4 R2cit 0.2 R2cit K9ac K14ac K18ac 0 T3phos n/a T3phos K9ac K14ac K18ac scale R2me2a T3phos R2me2a T3phos K9ac K14ac K18ac T6phos T6phos K9ac K14ac K18ac S10phos S10phos K9ac Interactions of acetyllysine- K9ac Albert Jeltsch marizing the interactions of K14ac (Back et al. 2011) bodies with acetylated pepti K18ac K9ac K14ac H3K14ac antibodies from th K9ac K18ac Jason Lieb/ModENCODE on a scale from 0 to 1 with 1 K14ac K18ac (Egelhofer et al 2010) e id K9ac K14ac K18ac H3K14ac pt pe abcam active millipore no motif Heat map summarizing the interactions of H3 peptides containing H3K4me3 in combinations with other H3 modifications. Interactions for two independent arrays are plotted on a scale from 0 to 1 with 1 (yellow) Equal amounts of yeast ex being the most significant. been preincubated with var µg/ml final concentration). shown for the negative cont
  21. 21. Influence of neighboring PTMs on effector protein interaction
  22. 22. H3K4me3-specific effector proteinsChromatin remodeling HATs/HDACs HMT HDMT JHDM3A Tudor TAF3 PHD
  23. 23. The histone PTM landscape can “fine-tune” the association of H3K4me3-effector proteins c c c 3a 3a 3a + + + e0 e3 e3 e0 e3 e3 e0 e3 e3 m m m m m m m m m K4 K4 K4 K4 K4 K4 K4 K4 K4 H3 H3 H3 H3 H3 H3 H3 H3 H3 – H3cit2 H3R2me1 H3R2me2a H3R2me2a/T3phos H3T3phos H3T6phos H3S10phos Rag2-PHD Chd1-Chromo BPTF-PHD+Bromo (Rag2) (CHD1) (BPTF in NURF) John Denu/Ben Garcia
  24. 24. Histone Code readers DDT PHD PHD bromo BPTF (NURF)PWWP Jmjc PHD PHD tudor tudor JMJD2A Jmjn (demethylase) bromo bromo bromo bromo bromo bromo BAH BAH HMG BAF180 (SWI/SNF) PHD PHD chromo chromo DEXD HELIC CHD4 (NuRD) (Figure from Abcam)
  25. 25. What are some other technologiesbeing used to ‘crack’ the histone code?
  26. 26. Mass spectrometry is a vital tool in combinatorial PTM discovery A. Bottom-up MS RP-HPLC Trypsin RP-HPLC H3 H3 MS ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQ DFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134 B. Top-down MS RP-HPLC HILIC H3 MS H3 (hydrophilic interaction liquid chromatography) ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQ DFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA-134
  27. 27. Mass spectrometry is a vital tool in combinatorial PTM discovery
  28. 28. SILAC-based approaches are unlocking identification of novel(Stable isotope labeling byamino acids in cell culture) effector proteins peptide Bead peptide Bead Isotope- Unlabeled labeled extracts extracts peptide Bead peptide Bead SDS/PAGE and tryptic digestion m/z
  29. 29. SILAC-based approaches are unlocking identification of novel effector proteins
  30. 30. Semi-synthetic modified nucleosomes explore multivalent engagements in chromatin thioester cysteine peptide ligation methyl aminoethylhalideNative chemical ligation (NCL) and Expressed protein ligation(EPL) (Kent/Cole/Muir labs) Methyl-lysine analogue (MLA) (Shokat et al.)
  31. 31. Semi-synthetic modified nucleosomes explore multivalent engagements in chromatin
  32. 32. Acknowledgments Strahl Lab Collaborations Andromeda Cook Cheryl Arrowsmith (SGC Toronto) * Raghu Dronamraju Mark Bedford (MD Anderson) * Deepak Jha Scott Briggs (Purdue) Stephen Fuchs * Stephen Frye (UNC) Jeff Jones Or Gozani (Stanford)Krzysztof Krajewski * Steve Jacobsen (UCLA) Jorge Martinez * Michael Keogh (Albert Einstein)Stephen McDaniel Shohei Koide (Univ. of Chicago) Julia Nepper Yang Shi (Harvard) Mike Parra Todd Stukenberg (UVa) Scott Rothbart * Ashutosh Tripathy (UNC)* Glenn Wozniak Marcey Waters (UNC) FundingNational Institutes of Health

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