[Keywords: p53 tumor suppressor protein; p21 gene; apoptosis; transcription elongation; RNA polymerase II; P-TEFb] Supplemental material is available at http://www.genesdev.org.

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.

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.[Keywords: HIV-1 Tat; CycT1:CDK9/P-TEFb; c-Ski-interacting protein; transcription elongation; alternative splicing; HIV-1 TAR RNA] Supplemental material is available at http://www.genesdev.org.

p53 is a pleiotropic transcription factor driving a flexible transcriptional program that mediates disparate cellular responses to stress, including cell cycle arrest and apoptosis. The mechanisms by which p53 differentially regulates its diverse target genes remain poorly understood. In this issue of Genes & Development, Morachis and colleagues (pp. 135–147) demonstrate the critical role of core promoter elements at p53 target loci, in that they dictate differential RNA polymerase II recruitment and activity in a p53-autonomous fashion.