Application of a Histone Peptide Array to IdentifyNovel 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
Purpose1. Determine if YEATS domain recognize Histonemodifications2. YEATS Domain proteins in Super ElongationComplex recognize Histone Modifications
Methods DNA Insertion into PGEX-Amplification 6P-1 Cloning Vector Transformation into competent DH5a Cells GST Protein Expression and Purification in competent Rosetta 2 cellsPeptide Microarray GST Peptide Pull-down Assay
DNA Amplification 1000 1000 400 400 100 100Coding sequences of YEATS domain were amplified byPCR using cDNA derived from HeLa cell lines. AF9 andENL coding sequences were successfully amplified.
Cloning Restriction Digest −+−+−+−+−+−+− 5,000 4,000 3,000 2,000 1,500 1,000 1,000 900 800 700 700 600 500 500 400 300 300 200AF9 and ENL were inserted into cloning vector PGEX-6P-1 (4900 bp) between restrictionsites BamHI and XhoI via ligation reaction. Vector containing inserts were transformed intoDH5a cells. YEATS domains constructs were created for plasmids containing inserts AF9 orENL.
GST Protein Expression and Purification ENL AF9 70 55 35 25Samples AF9 and ENL were expressed and purified by GST-Tagging. Both GST-tagged proteins AF9 and ENL are 40 KDin weight.
Peptide Microarray ENL-Y 500gainA peptide microarray platform was utilized to screen candidatesites of recognition by YEATS motif member ENL.ENL appears to show strong association with Histone 4.
GST Peptide Pull-Down Assay ENL AF9 JMJD2A H4K20 H4K20 H4K20 Input me0 me1 me2me3 Input me0 me1 me2 me3 Input me0me1me2me355 55 5535 35 3525 25 25 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 ElongationComplex to release RNA Polymerase II from promoter region toelongation stage for HOX gene expression. Future studies areneeded to determine this pathway.SEC Model by Lin et. al 2003
Acknowledgements CPRIT Summer Undergraduate program Dr. Xiaobing Shi Members of Shi Laboratory