Gene-specific repression of the p53 target gene PUMA via intragenic CTCF–Cohesin binding

Abstract

The p53 transcriptional program orchestrates alternative responses to stress, including cell cycle arrest and apoptosis, but the mechanism of cell fate choice upon p53 activation is not fully understood. Here we report that PUMA (p53 up-regulated modulator of apoptosis), a key mediator of p53-dependent cell death, is regulated by a noncanonical, gene-specific mechanism. Using chromatin immunoprecipitation assays, we found that the first half of the PUMA locus (~6 kb) is constitutively occupied by RNA polymerase II and general transcription factors regardless of p53 activity. Using various RNA analyses, we found that this region is constitutively transcribed to generate a long unprocessed RNA with no known coding capacity. This permissive intragenic domain is constrained by sharp chromatin boundaries, as illustrated by histone marks of active transcription (histone H3 Lys9 trimethylation [H3K4me3] and H3K9 acetylation [H3K9Ac]) that precipitously transition into repressive marks (H3K9me3). Interestingly, the insulator protein CTCF (CCCTC-binding factor) and the Cohesin complex occupy these intragenic chromatin boundaries. CTCF knockdown leads to increased basal expression of PUMA concomitant with a reduction in chromatin boundary signatures. Importantly, derepression of PUMA upon CTCF depletion occurs without p53 activation or activation of other p53 target genes. Therefore, CTCF plays a pivotal role in dampening the p53 apoptotic response by acting as a gene-specific repressor.[Keywords: p53; PUMA; CTCF; cohesin; apoptosis; noncoding RNA] Supplemental material is available at http://www.genesdev.org.

Genome-wide targeting of the epigenetic regulatory protein CTCF to gene promoters by the transcription factor TFII-I

“…This data also suggests that the position of CTCF binding relative to the transcription start site may greatly influence its role in the transcriptional process. Our data indicates proximal promoter bound CTCF acts as an activator of initiation, whereas other reports have demonstrated CTCF bound within exonic regions acts as a negative regulator of transcription through enhanced pausing (9,45). Because of the link between TFII-I and genes involved in metabolism, we wanted to test TFII-I KD cells for an altered response to nutrient deprivation.…”

The histone acetyltransferase PCAF regulates p21 transcription through stress-induced acetylation of histone H3

“…Previous analyses of the kinetics of p21 transcriptional activation suggest that histone acetylation is induced following a multitude of genotoxic stresses, but that histone acetylation alone is not sufficient to promote transcriptional elongation by poised Pol II. [49][50][51] This suggests that acetylation of A2066 (Sigma Aldrich), p14 ARF DCS-241 (Santa Cruz), p300 N-15 (Santa Cruz), CBP A-22 (Santa Cruz). When used, image brightness and contrast processing (ImageJ) was applied to the entire blot, maintaining linear exposures.…”

De-SUMOylation of CCCTC Binding Factor (CTCF) in Hypoxic Stress-induced Human Corneal Epithelial Cells

“…Therefore, CTCF must be capable of dynamically responding to stresses and mediating stress-induced alteration of chromatin structures. Several stress-related proteins such as PUMA (p53 upregulated modulator of apoptosis) have been reported to be regulated by CTCF (26). However, it is still unclear how CTCF is regulated in response to environmental stresses.…”