Gene-specific requirement for P-TEFb activity and RNA polymerase II phosphorylation within the p53 transcriptional program

Activation of the p53 pathway mediates cellular responses to diverse forms of stress. Here we report that the p53 target gene p21^CIP1 is regulated by stress at post-initiation steps through conversion of paused RNA polymerase II (RNAP II) into an elongating form. High-resolution chromatin immunoprecipitation assays (ChIP) demonstrate that p53-dependent activation of p21^CIP1 transcription after DNA damage occurs concomitantly with changes in RNAP II phosphorylation status and recruitment of the elongation factors DSIF (DRB Sensitivity-Inducing Factor), P-TEFb (Positive Transcription Elongation Factor b), TFIIH, TFIIF, and FACT (Facilitates Chromatin Transcription) to distinct regions of the p21^CIP1 locus. Paradoxically, pharmacological inhibition of P-TEFb leads to global inhibition of mRNA synthesis but activation of the p53 pathway through p53 accumulation, expression of specific p53 target genes, and p53-dependent apoptosis. ChIP analyses of p21^CIP1 activation in the absence of functional P-TEFb reveals the existence of two distinct kinases that phosphorylate Ser5 of the RNAP II C-terminal domain (CTD). Importantly, CTD phosphorylation at Ser2 is not required for p21^CIP1 transcription, mRNA cleavage, or polyadenylation. Furthermore, recruitment of FACT requires CTD kinases, yet FACT is dispensable for p21^CIP1 expression. Thus, select genes within the p53 pathway bypass the requirement for P-TEFb and RNAP II phosphorylation to trigger a cellular response to inhibition of global mRNA synthesis.