Identificaton and Characterization of Histone
Lysine Decrotonylases
Dr. Xiang David Li, Department of Chemistry
Background: In the nuclei of all eukaryotic cells, genomic DNA is highly compacted into chromatin by wrapping around ‘spools’, which are comprised of histone proteins. Diverse posttranslational modifications (e.g., acetylation, methylation and phosphorylation) of histones are known to regulate
a variety of fundamental cellular processes, such as gene transcription, DNA replication and DNA damage repair. As a result, errors in cellular control of
these histone PTMs are often associated with human diseases such as cancer. Therefore, it is important to understand how a histone PTM is regulated in
cells. Lysine crotonylation (Kcr) is a newly identified histone PTM that has been found to enrich at active gene promoters and potential enhancers in
mammalian cell genomes. However, the cellular functions of Kcr still remain elusive, which is, in large part, due to a lack of knowledge about enzymes
that regulate the addition or removal of this new PTM.
Objective
2:
To
analyze
how
histone
crotonylation
is
regulated
by
Objective 1: To identify enzymes that ‘erase’ lysine crotonylation ‘marks’
histone
decrotonylases.
on histones.
Strategy:
Crystal structure analysis of Sirt3-H3K4Cr complex
LC-MS based enzyme activity assay
Chromatin immunoprecipitation (ChIP)-PCR and RNA-PCR
Kcr is enriched at active gene promoters and potential enhancers in mammalian cell genomes, while the
regulating enzymes are unknown.
Challenge: Involved in weak or transient protein-protein interactions
Strategy:
Photo-reactive chemical probes to capture ‘erasers’ and ‘writers’
SILAC-based quantitative proteomics for comprehensive identification.
Crystallization analysis
Chromatin immunoprecipitation (ChIP)PCR or Seq
Enzyme activity assay
Results: We found that Sirt1, Sirt2 and Sirt3 can catalyze the hydrolysis
of lysine crotonylated histone peptides and proteins in vitro in an NADdependent manner. More importantly, Sirt3 functions as a decrotonylase
to regulate histone Kcr dynamics and gene transcription in living cells.
Photo-reactive chemical probes to capture ‘erasers’ and ‘writers’.
16
8
0
19
20
21
22
20
21
22
20
21
22
14
7
SILAC-based quantitative proteomics for comprehensive identification
0
19
Results: We use a chemical proteomics approach to comprehensively profile ‘eraser’ enzymes that recognize a lysine-4 crotonylated histone H3
(H3K4Cr) mark. We found that Sirt1, Sirt2 and Sirt3 could be selectively
captured by H3K4Cr mark and Sirt3 showed the highest affinity.
Time (min)
0
Sirt3
cal/sec
0.5
Sirt2
0.0
100
40
50
50
0
-1
25
1
10
100
1000
Sirt3 catalyzes the hydrolysis of lysine crotonylated histone
peptides and proteins in vitro in an NAD-dependent manner
and the endogenous Sirt3 catalyzes decrotonylation of histones in living cells
-1.00
Lysine crotonylation ChIP-qPCR
control siRNA
0.00
-2.00
-4.00
-6.00
-8.00
*
20
% of input
4
The binding pocket formed by hydrophobic residues that accommodate the crotonyl lysine, in which a  stacking
formed between residue Phe180 of Sirt3
and the crotonyl group.
25
75
kcal mol of injectant
2
30
0
19
-2.00
-0.5
0
20
0.00
Sirt1
-2
10
6
0.0 0.5 1.0 1.5 2.0 2.5
**
**
*
15
Sirt3 siRNA
*
10
5
Molar ratio
0
Identification of Sirt3 as a selective and tight binding partner of lysine-4 crotonylated histone H3 via CLASPI
approach. The recombinant Sirt3 was selectively labeled in vitro by crotonylated probe 1 and the labeling
by probe 1 was inhibited by a H3K4Cr peptide.
Publications
1. X. C. Bao, Y. Wang, X. Li, X. M. Li, Z. Liu, T. P. Yang,C. F. Wong, J. W. Zhang, Q. Hao, X. D.
Li*, Identification of 'erasers' for lysine crotonylated histone marks using a chemical
proteomics approach. eLife 2014, 3
2. T. P. Yang, X. M. Li, X. C. Bao, Y. M. Fung, X. D. Li*. Photo-lysine captures proteins that
bind lysine post-translational modifications. Nat Chem Biol 2016, 12(2), 70-2
3. T. P. Yang, Z. Liu, X. D. Li*, Developing diazirine-based chemical probes to identify histone modification 'readers' and 'erasers'. Chem Sci 2015, 6 (2), 1011-1017
Baz2a
Brip1
Corin
Ptk2
Tshz3
Wapal
Zfat
Relative mRNA level (/control siRNA)
1.0
12
2.5
control siRNA
Sirt3 siRNA
***
2
1.5
**
**
1
0.5
0
Baz2a
Brip1
Corin
Ptk2
Tshz3
Wapal
Zfat
Sirt3
ChIP-qPCR analyses revealed increased histone lysine crotonylation level of certain chromatin loci and upregulated Ptk2, Tshz3 and Wapal gene expression upon Sirt3 knockdown.
Conclusion and impact: The completion of these studies will provide
new opportunities for examining the physiological significance of histone
lysine crotonylation and provide critical information to help dissect its regulatory networks in chromatin biology and unravel unknown cellular mechanisms controlled by Sirt3. In the long-term, findings of this research may
also become the basis for identifying therapeutic targets for human diseases associated with misregulation of this histone PTM.