p53-Dependent p21 mRNA elongation is impaired when DNA replication is stalled.
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.
Mattia
Melissa
M
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Gottifredi
Vanesa
V
McKinney
Kristine
K
Prives
Carol
C
D000107:Acetylation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D003630:Daunorubicin / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012319:RNA Polymerase II / Q000378:metabolism; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D006657:Histones; D012333:RNA, Messenger; D035181:TATA-Box Binding Protein; D016159:Tumor Suppressor Protein p53; D012319:RNA Polymerase II; D006918:Hydroxyurea; D003630:Daunorubicin