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

1. BioTechniques Symposium: The Genome and Beyond
Understanding 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. Transcriptional regulation
Set2 Rad6 Set1
(H3-K36) (H2B-ub) (H3-K4)

3. Organization of eukaryotic chromatin
DNA double helix
Histone Nucleosomes
H3
H2B Solenoid
H2A
H4
Chromatin loop:
~100,000 bp DNA
Chromatin

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. 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. Histone Modifications
Acetylation
Phosphorylation
Methylation Histone Code
Chromatin
regulator
ADP-ribosylation
Ubiquitination
Sumoylation
Tail Globular

7. Epigenetic ‘Toolkit’
Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language
where context matters. J. Mol. Biol. 409:36-46.

8. Histone Modifications
Acetylation
Phosphorylation
Methylation Histone Code
Chromatin
regulator
ADP-ribosylation
Ubiquitination
Sumoylation
Tail Globular

9. Bromodomain-containing proteins can bind to acetylated histones!
(TBP)
TATAA
(adapted from E. Pennisi - Science, 2000)

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. Mechanisms of histone recognition
Gardner, Allis & Strahl (2011) OPERating ON chromatin, a colorful language
where context matters. J. Mol. Biol. 409:36-46.

12. Peptide microarrays

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
ac
ac-I A Q D F K T D L R F - Peg - K(biotin)-NH2 H3(74-84)
phos ac ac ac ac
ac-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 me2
ac-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. Design of our histone peptide arrays
Thanks to…
Or Gozani

15. Are histone modification-specific
antibodies really, um, specific?

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. 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. Are histone antibodies influenced
by neighboring PTMs?

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. 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. Influence of neighboring PTMs on
effector protein interaction

22. H3K4me3-specific effector proteins
Chromatin remodeling HATs/HDACs HMT HDMT
JHDM3A
Tudor
TAF3
PHD

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. 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. What are some other technologies
being used to ‘crack’ the histone code?

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. Mass spectrometry is a vital tool in combinatorial PTM discovery

28. SILAC-based approaches are unlocking identification of novel
(Stable isotope labeling by
amino 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. SILAC-based approaches are unlocking identification of novel
effector proteins

30. Semi-synthetic modified nucleosomes explore multivalent
engagements in chromatin
thioester cysteine
peptide ligation
methyl aminoethylhalide
Native chemical ligation (NCL) and
Expressed protein ligation(EPL)
(Kent/Cole/Muir labs)
Methyl-lysine analogue (MLA)
(Shokat et al.)

31. Semi-synthetic modified nucleosomes explore multivalent
engagements in chromatin

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)
Funding
National Institutes
of Health