ROLE OF ARG DEMETHYLATION IN REGULATING P53 PATHWAY

Open Access
Author:
Wang, Danchen
Graduate Program:
Cell and Molecular Biology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
April 05, 2010
Committee Members:
  • Yanming Wang, Thesis Advisor
Keywords:
  • JMJD4/6
  • PAD4
  • Arg demethylation
  • p53
Abstract:
Post-translational histone modifications control transcription by regulating higher order chromatin structures and access to the underlying DNA. Previous studies have shown that histone Arg methylation and Lys acetylation coordinate to regulate the expression of p53-target genes. However, whether Arg demethylation also plays a role in regulating p53 pathway is largely unknown. PAD4 catalyzed histone deimination and demethylimination could turn Arg and monomethyl-Arg into citrulline, respectively. In my thesis, I have studied the function of PAD4 in the formation of a decondensed chromatin structure called neutrophil extracellular traps (NETs) and analyzed the interaction between PAD4 and HDAC2 by GST-pull down and re-CHIP experiments. Besides histone demethylimination by PAD4, Jumonji domain-containing 6 (JMJD6) was reported to be an Arg demethylase. To investigate the function of JMJD6 and its homolog JMJD4, I have established Flag-JMJD6 and Falg-JMJD4 expressing cell lines, purified the JMJD4 and JMJD6 fusion proteins and tested their demethylation function both in vivo and in vitro. Further, I depleted JMJD4 and JMJD6 by shRNA or siRNA in U2OS cells and tested the changes of p53-target gene expression. Through my thesis study, I found that the formation of NETs is associated with PAD4 activation and global histone citrullination. I found that PAD4 and HDAC2 coordinate with each other and act as p53 corepressors. My study also showed that the JMJD4 and JMJD6 both have weak but detectable demethylation activity and bind to p53 to regulate the expression of p53-target genes. Additionally, I analyzed the newly synthesized derivatives of PAD4 inhibitor Cl-amindine and found several candidates with improved PAD4 inhibition activity.