Citrullination of Inhibitor of Growth 4 (ING4) by Peptidylarginine Deminase 4 (PAD4) Disrupts the Interaction between ING4 and p53

Abstract

Gene expression is regulated by a number of interrelated posttranslational modifications of histones, including citrullination. For example, peptidylarginine deminase 4 (PAD4) converts peptidyl arginine to citrulline in histone H3 and can repress gene expression. However, regulation of gene expression through citrullination of non-histone proteins is less well defined. Herein, we identify a tumor suppressor protein, inhibitor of growth 4 (ING4), as a novel non-histone substrate of PAD4. ING4 is known to bind p53 via its nuclear localization signal (NLS) region and to enhance transcriptional activity of p53. We show that PAD4 preferentially citrullinates ING4 in the same NLS region and thereby disrupts the interaction between ING4 and p53. A citrulline-mimicking Arg-NLS-Gln ING4 mutant, which has all Arg residues in the NLS mutated to Gln, loses its affinity for p53, can no longer promote p53 acetylation, and results in repression of downstream p21 expression. In addition, we found that citrullination leads to increased susceptibility of ING4 to degradation, likely impacting p53-independent pathways as well. These findings elucidate an interaction between posttranslational citrullination, acetylation, and methylation and highlight an unusual mechanism whereby citrullination of a non-histone protein impacts gene regulation. Gene expression is regulated by a number of interrelated posttranslational modifications of histones, including citrullination. For example, peptidylarginine deminase 4 (PAD4) converts peptidyl arginine to citrulline in histone H3 and can repress gene expression. However, regulation of gene expression through citrullination of non-histone proteins is less well defined. Herein, we identify a tumor suppressor protein, inhibitor of growth 4 (ING4), as a novel non-histone substrate of PAD4. ING4 is known to bind p53 via its nuclear localization signal (NLS) region and to enhance transcriptional activity of p53. We show that PAD4 preferentially citrullinates ING4 in the same NLS region and thereby disrupts the interaction between ING4 and p53. A citrulline-mimicking Arg-NLS-Gln ING4 mutant, which has all Arg residues in the NLS mutated to Gln, loses its affinity for p53, can no longer promote p53 acetylation, and results in repression of downstream p21 expression. In addition, we found that citrullination leads to increased susceptibility of ING4 to degradation, likely impacting p53-independent pathways as well. These findings elucidate an interaction between posttranslational citrullination, acetylation, and methylation and highlight an unusual mechanism whereby citrullination of a non-histone protein impacts gene regulation.