Novel DNA damage induced pathways to cancer: the role of PCAF histone acetyltransferase

Abstract

Cell cycle checkpoints act as surveillance system and following DNA damage caused by ultraviolet (UV) or ionising irradiation (IR), ATM and ATR phosphorylate various downstream substrates that preserve the genome integrity. The tumor suppressor protein p53 is a target for ATM/ATR kinases and modulates the expression of target genes involved in DNA damage response and promotes DNA repair, cell cycle arrest, and apoptosis. ATM/ATR and Chk2 phosphorylates p53 at Ser15 and Ser20 respectively. PCAF belongs to histone acetyltranferases (HATs) family and they acetylate nucleosomal and free histones as well as non-histone proteins such as p53. PCAF acetylates p53 at Lys320 which enhance p53 binding affinity to target genes and increasing p53 mediated transcriptional activity respectively. Our hypothesis is that the ATM/ATR induced pathway might modulates PCAF activation since PCAF protein sequence has shown to contain four putative ATM/ATR target sites as well as two Chk1/Chk2 sites. Also, p53 contributes in PCAF regulation.Our results show that PCAF regulation is cell specific and different cellular stresses introduce different post-translational modifications on PCAF. p21 is known to be targeted by p53 and PCAF following DNA damage, and our results have shown an elevation in p21 mRNA levels in U2OS (p52+/+) cells and a decrease in Saos-2 (p53-/-) and pEBS7(ATM-/-) cells following etoposide treatment, which suggests that both ATM and p53 are required for PCAF to be fully activated in ATM induced pathway. PCAF and PCAF k-r mutant, which lack the auto-acetylation region, were over-expressed and assessed under different cellular stresses by measuring the p21 tagged promoter for luciferase (p21 luc) and p21 mRNA levels. Following UV treatment, both the p21 mRNA and p21 luc levels were highly elevated in PCAF k-r over-expression in U2OS cells. Also, PCAF defective mutant, PCAF∆HAT, has shown different impact on PCAF activity under different genetic insults in comparison to PCAF k-r mutant. . . Our findings suggest that ATM, ATR induced pathways andChk2 kinase negatively regulate PCAF activity since the co-expression of the latter kinases with PCAF, downregulated PCAF proteins abundance and p21-luc levels in etoposide treated cells following the over-expression of PCAF. In UV induced pathway, Chk1 kinase seems to have a positive effect on PCAF as p21 luc promoter levels were downregulated in U2OS cells co-expressed with Chk1 kinase dead mutant. Our chromatin immunoprecipitation data indicates that p53 is highly recruited on PCAF's own promoter in U2OS cells following etoposide treatment, which suggests a regulatory role of p53 on PCAF regulation. Moreover, the recruitment of PCAF and p53 on p21 promoter seems to be determined by the nature of the damage signal.

Date of Award	31 Dec 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor) & Marija Krstic-Demonacos (Supervisor)