p53-Dependent p21 mRNA Elongation Is Impaired when DNA Replication Is Stalled

Abstract

We have previously reported that when DNA replication is blocked in some human cell lines, p53 is impaired in its ability to induce a subset of its key target genes, including p21 WAF1/CIP1. Here, we investigated the reason for this impairment by comparing the effects of two agents, hydroxyurea (HU), which arrests cells in early S phase and impairs induction of p21, and daunorubicin, which causes a G 2 block and leads to robust activation of p21 by p53. HU treatment was shown to inhibit p21 mRNA transcription rather than alter its mRNA stability. Nevertheless, chromatin immunoprecipitation assays revealed that HU impacts neither p53 binding nor acetylation of histones H3 and H4 within the p21 promoter. Furthermore, recruitment of the TFIID/TATA-binding protein complex and the large subunit of RNA polymerase II (RNA Pol II) are equivalent after HU and daunorubicin treatments. Relative to daunorubicin treatment, however, transcription elongation of the p21 gene is significantly impaired in cells treated with HU, as evidenced by reduced occupancy of RNA Pol II at regions downstream of the start site. Likewise, in the p21 downstream region after administration of HU, there is less of a specifically phosphorylated form of RNA Pol II (Pol II-C-terminal domain serine 2P) which occurs only when the polymerase is elongating RNA. We propose that while the DNA replication checkpoint is unlikely to regulate the assembly of a p21 promoter initiation complex, it signals to one or more factors involved in the process of transcriptional elongation.

Mechanisms of regulatory diversity within the p53 transcriptional network

“…Identical stimulus-specific recruitment of transcriptional coregulators is observed at the Mdm2 promoter, but not at the Fas promoter, revealing the existence of both stimulus-and promoterspecific events. Work from the Prives lab has generated similar observations (Mattia et al, 2007). In this case, Mattia et al analysed the p21 waf1 promoter in cells treated with daunorubicin (high p21 waf1 transcription) versus hydroxyurea (HU) (low p21 waf1 transcription).…”

“…Once again, p53 binding and p53-mediated histone acetylation was identical in both scenarios. Importantly, Pig3 did not show this mode of stimulus-specific regulation (Mattia et al, 2007).…”

“…In this case, Mattia et al analysed the p21 waf1 promoter in cells treated with daunorubicin (high p21 waf1 transcription) versus hydroxyurea (HU) (low p21 waf1 transcription). Strikingly, ChIP analysis in these two scenarios revealed that RNAP II activity was differentially regulated at post-initiation steps, with a blockage of RNAP II elongation being evident only in HU-treated cells (Mattia et al, 2007). Once again, p53 binding and p53-mediated histone acetylation was identical in both scenarios.…”

Transcriptional Regulation by P53

“…behavior under similar conditions (Donner et al 2007;Mattia et al 2007). These different stresses likely lead to the activation of different signaling cascades, thus altering the availability of key stimulus-specific coregulators-such as components of Mediator, GTFs, etc.-that are necessary for the transactivation of a certain locus.…”

“…However, for many genes, such as p21, it is clear that the levels of p53 bound are not the sole determinant of the ensuing transcriptional response (Espinosa et al 2003;Donner et al 2007;Mattia et al 2007;Hill et al 2008b). In fact, it appears that similar levels of bound p53 can direct a differential PIC assembly that is both locus-and stimulus-specific (Espinosa 2008), as depicted in Figure 3.…”