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