Abstract LB-85: Damage-inducible intragenic demethylation activates transcription of an alternative intronic promoter in TP53-wildtype but not mutant human cells and tumors

Abstract

Abstract Unlike the well-known effects of promoter methylation, the functional significance of altered intragenic (gene body) CpG methylation remains unclear, particularly in genes such as the TP53 tumor suppressor which lack a 5′ CpG island. Here we show that CpG-specific demethylation of TP53 exon 5 in normal human and mouse cells is inducible by X-irradiation; this is accompanied by co-expression of a 5′-truncated TP53 isoform, raising the possibility that demethylation permits transcription from the known alternative promoter (P2) in intron 4. Consistent with this, in vivo expression of fully pre-methylated TP53 exonic constructs is associated with reduced basal and damage-inducible expression of both full-length and truncated TP53. In contrast, virally TP53-reconstituted Caco-2 and PC-3 human cancer cell lines respond to damage by upregulating expression of full-length TP53 and reducing expression of the truncated RNA isoform; CDKN1A expression varies directly with P2 isoform expression but inversely with the P1 transcript in these cells, suggesting that the isoform mediates downstream transactivation. 450K methylation analysis of adjacent normal and TP53-mutated human pancreatic cancer tissues suggests a mutation-specific decrease of intragenic TP53 methylation in intron 1 near P1 (p < 0.02 ) and a reciprocal increase in intron 4 near P2 (p < 0.01). Archival TP53 database analysis indicates that CpG-specific mutation frequency in human tumors is inversely associated with damage-inducible demethylation at the same sites in vitro (Spearman coefficient -7.1 ), consistent with methylation-dependent downregulation of transcription-coupled repair. We conclude that intragenic alterations of TP53 methylation are both physiologically inducible and functional, and hypothesise that tumor progression is drivable by either (i) intragenic hypermethylation affecting P1 as a field defect in TP53-wt tumor or adjacent normal tissues, or (ii) damage-inducible repression of P2 isoform expression associated with defective intron 4 demethylation, together with overexpression of full-length p53, in TP53-mutant tumors and cells. Citation Format: James Blackburn, Robert Ng, Daniel Roden, Jianmin Wu, Richard J. Epstein. Damage-inducible intragenic demethylation activates transcription of an alternative intronic promoter in TP53-wildtype but not mutant human cells and tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-85. doi:10.1158/1538-7445.AM2014-LB-85