Post on 30-Oct-2014
description
Akhil Sood, College Student, Emory University, Atlanta, GAShi Lab, MD Anderson Cancer Center, Houston, TX
Application of a Histone Peptide Array to Identify Novel Reader Proteins of Histone
Modifications
Introduction
Eukaryotic DNA is wrapped around histone proteins to form chromatin
Chromatin modifications influence many biological processes
Histone Modifications
Chromatin structure regulated by Post Translational Modifications (PTMs) of Histones
Acetylation Methylation Phosphorylation Ubiquitination
Histone Code- histone modifications determine biological outcome
Reader Domains
“Reader” Domains- conserved domains that bind and recognize histone modifications
Interpret modification to carry out biological function
Bromodomains- acetyl
Chromodomains- methyl
PHD Fingers- tri-methyl Kouzarides, Cell 2007
YEATS Domain
Role in Chromatin Modification not well known
Highly conserved domain found in many proteins and organisms
YEATS domain in Humans (AF9, ENL, GAS41, and YETS2) linked to transcription, DNA repair, and cancer
No common function found among YEATS members
Super Elongation Complex
AF9 and ENL members of SEC
Super Elongation Complex regulates transcription elongation of HOX Genes in development
Releases paused Pol II from promoter site to elongation stage for HOX Gene Expression
Role of AF9 and ENL in SEC unknown
Purpose
1. Determine if YEATS domain recognize Histone modifications
2. YEATS Domain proteins in Super Elongation Complex recognize Histone Modifications
Methods
DNA Amplification
Insertion into PGEX-6P-1 Cloning Vector
Transformation into competent DH5a
Cells
GST Protein Expression and Purification in
competent Rosetta 2 cells
Peptide Microarray GST Peptide Pull-down Assay
DNA Amplification
Control
AF9
ENL
GAS41
YETS2
100
400
1000
100
400
1000
Coding sequences of YEATS domain were amplified by PCR using cDNA derived from HeLa cell lines. AF9 and ENL coding sequences were successfully amplified.
Cloning
300
500
700
1,000
1,5002,0003,0004,0005,000
200300
4005006007008009001,000
1. 1
kb L
adder
2. p
GEX
-6P-1
3. E
NL-
PGEX
(A)
4. E
NL-
PGEX
( A
)
5. E
NL-
PGEX
( B
)
6. E
NL-
PGEX
(B)
7. E
NL-
PGEX
(C)
8. E
NL-
PGEX
(C)
9. A
F9-P
GEX
(A)
10. AF9
-PGEX
(A)
11. AF9
-PGEX
(B)
12. AF9
-PGEX
(B)
13. AF9
-PGEX
(C)
14. AF9
-PGEX
( C
)
15. 10
0bp L
adder
Restriction Digest − + − + − + − + − + − + −
AF9 and ENL were inserted into cloning vector PGEX-6P-1 (4900 bp) between restriction sites BamHI and XhoI via ligation reaction. Vector containing inserts were transformed into DH5a cells. YEATS domains constructs were created for plasmids containing inserts AF9 or ENL.
GST Protein Expression and Purification
Samples AF9 and ENL were expressed and purified by GST-Tagging. Both GST-tagged proteins AF9 and ENL are 40 KD in weight.
35
25
5570
ENL AF9
Peptide Microarray
A peptide microarray platform was utilized to screen candidate sites of recognition by YEATS motif member ENL. ENL appears to show strong association with Histone 4.
ENL-Y 500gain
GST Peptide Pull-Down Assay
Input me1me2me3
25
35
55
H4K20me0
25
35
55
H4K20Input me0 me1 me2 me3
25
35
55
Input me0me1me2me3H4K20
AF9 ENL JMJD2A
GST peptide pull-down assays were performed to determine sites of recognition by YEATS motif. Compared to control sample JMJD2A, Western analysis shows that both AF9 and ENL are not methyl-specific.
Conclusion
AF9 and ENL were cloned, expressed, and purified
AF9 and ENL bind to histone 4 tail, independent of modifications
Not methyl-specific for recognition
Future studies necessary to determine role of YEATS domain in other histone modifications.
Histone tail binding likely mechanism for regulating Super Elongation Complex
Working Model: AF9 and ENL bind to histone 4 tailH4 tail binding by AF9/ENL can stimulate the Super Elongation Complex to release RNA Polymerase II from promoter region to elongation stage for HOX gene expression. Future studies are needed to determine this pathway.
SEC Model by Lin et. al 2003
Acknowledgements
CPRIT Summer Undergraduate program
Dr. Xiaobing Shi
Members of Shi Laboratory