A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint

Abstract

We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 39 end of the p21 gene. Recruitment of specific elongation and 39 processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/ 39 processing machinery.[Keywords: Chk1; Chk2; S-phase checkpoint; p53; transcription elongation] Supplemental material is available at http://www.genesdev.org.

Transcriptional Regulation by P53

“…Furthermore, select basal components of the transcription initiation machinery are recruited to certain p53 target genes such as p21 regardless of the nature of the stimulus, whereas the assembly of other factors is strongly dependent on the nature of the stress. For example, both TFIIB and cdk8 are recruited to p21 after DNA damage or nutlin treatment, but they are absent after treatment with more mild forms of stress such as hydroxyurea or UVC (Espinosa et al 2003;Donner et al 2007;Beckerman et al 2009); the recruitment of these two factors correlates well with transactivation of p21 RNA. In contrast, other cofactors such as TBP or MED1 show no such differential .…”

“…It is not surprising, then, that several recent studies have documented a role for p53 and its upstream signaling pathways in the stimulusand locus-specific control of transcription elongation (Gomes et al 2006;Donner et al 2007;Mattia et al 2007;Hill et al 2008a ). In the case of chromium exposure, the elongation of p21 but not puma RNA is inhibited, in a process that implicates DNA-PK (Hill et al 2008b), whereas after replication stress, p21 elongation is inhibited in a Chk1-dependent fashion (Beckerman et al 2009). Further work is required to extend these observations to other p53 targets, as the regulation of transcription elongation is emerging as a key layer of control in the fine-tuning of the p53 response.…”

Dual association by TFAP2A during activation of the p21cip/CDKN1A promoter

“…4A and C). Of note, the block in CDKN1A transcriptional elongation observed in some colon carcinoma lines upon HU treatment 20 was not apparent under our experimental conditions. However, we did also observe a significant induction of TFAP2A mRNA and protein in treated cells (Fig.…”

p53-dependent Induction of PVT1 and miR-1204

“…Chromatin Immunoprecipitation Analysis-ChIP analysis was carried out as described previously (36). Briefly, p53 was immunoprecipitated with monoclonal antibodies DO1 and 1801.…”