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P53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation.

ABSTRACT: It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.

SUBMITTER: Laptenko O

PROVIDER: S-EPMC3127872 | biostudies-other | 2011 Jun

REPOSITORIES: biostudies-other

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p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation.

Laptenko Oleg O Beckerman Rachel R Freulich Ella E Prives Carol C

Proceedings of the National Academy of Sciences of the United States of America 20110523 26

It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupanc ...[more]

PMID: 21606339

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Project description:BackgroundSmall proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.ResultsUsing a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.ConclusionUp-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.

Project description:DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes involved in cell cycle control and cell survival.

Dataset Information

P53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation.

Publications

p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation.

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