The Enzymatic Properties of Peptidylarginine Deiminase 4 and its Negative Regulatory Effect Upon NSAID Activated Gene-1

Open Access
Doret, Patrick Thomas
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Master of Science
Document Type:
Master Thesis
Date of Defense:
April 02, 2008
Committee Members:
  • Yanming Wang, Thesis Advisor
  • NSAID Activated Gene-1
  • Peptidylarginine Deiminase 4
  • Citrullination
The repressive structure of chromatin is subject to a diverse collection of post-translational histone modifications which control transcription by regulating access to the underlying DNA. Combinations of epigenetic modifications affect gene expression through the ¡¥histone code¡¦ in a synergistic or antagonistic fashion. These modifications are dynamically regulated allowing chromatin to transition between transcriptionally active or silent states. Thus, epigenetic marks arranged on chromatin function as a fundamental regulatory mechanism to control eukaryotic transcription. Numerous residues located on histones are covalently modified by regulatory enzymes. Specifically, arginine residues located on core histone N-terminal tails may be mono-methylated, asymmetrically or symmetrically di-methylated. These arginine isoforms affect gene expression through two possible consequences: creation of possible binding sites for proteins and disruption of potential hydrogen bonding. Thus, methylated arginine residues function as crucial epigenetic marks. Throughout this study, the histone modifying enzyme protein arginine deiminase 4 (PAD4), which catalyzes the deimination or demethylimination of arginine and mono-methyl arginine, was examined. PAD4 dynamically regulates arginine residues by producing citrulline, an unconventional amino acid. Although PAD4 modifies both arginine and mono-methyl arginine, there has been no evidence supporting that PAD4 regulates the di-methyl form. My studies present data indicating PAD4 may catalyze the ¡¥demethylation¡¦ of di-methyl arginine through a novel regulatory mechanism. Furthermore, two compounds were used to study the repressive effects of histone citrullination on gene expression. The first compound, Cl-amidine, irreversibly inhibits PAD4 activity thus preventing the dynamic regulation of arginine residues. The second compound, resveratrol, increases expression of p53 target genes. MCF-7 cells treated with either compound displayed elevated expression of the TGF-ƒÒ family member NSAID activated gene-1 (NAG-1). Chromatin immunoprecipitation following treatment with resveratrol revealed reduced levels of histone citrullination and increased levels of histone arginine methylation. Together, these studies indicate that PAD4 negatively regulates the transcription of NAG-1 through citrullination of the promoter nucleosomes. MCF-7 cells treated with either compound showed reduced levels of growth, a possible downstream effect of the up-regulated tumor suppressor NAG-1. Following translation and secretion, this protein has been postulated to function through membrane bound receptors. Immunostaining against NAG-1 showed increased translocation to the cellular membranes after resveratrol treatment, indicating the protein may be inducing apoptosis. Thus, the inhibition of proliferating MCF-7 cells by resveratrol or Cl-amidine may be, in part, due to the increased expression of NAG-1.