CBP and p300 Histone Acetyltransferases Contribute to Homologous Recombination by Transcriptionally Activating the BRCA1 and RAD51 Genes

Hideaki Ogiwara,Takashi Kohno,Sue Cotterill

doi:10.1371/journal.pone.0052810

Abstract

Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

Highlights

Chromosomal DNA and histones form a highly condensed structure known as chromatin
The possible involvement of CBP and p300 in double-strand break (DSB)-induced homologous recombination (HR) was investigated in vivo by using a GFP-based chromosomal assay in which a DSB is generated by expression of the I-SceI endonuclease, whose recognition site is integrated in the GFP gene such that digestion disrupts the gene (Figure 1A) [19]
Quantitative PCR analysis showed that enrichment of CBP and p300 at the BRCA1 and RAD51 promoter regions, which contain E2F transcription factor binding sites [17], was higher than at the GAPDH gene body region, i.e., a unrelated gene region (Figure 3A–B). These results indicated that CBP and p300 directly bind to the BRCA1 and RAD51 promoter regions

Summary

Introduction

Chromosomal DNA and histones form a highly condensed structure known as chromatin. Access to chromosomal DNA by transcription factors and DNA repair proteins is regulated by chromatin remodeling, including histone modification and nucleosomal alteration [1]. We recently reported that the homologous HATs CBP and p300 facilitate DSB repair by acetylating histones H3 and H4 at DSB sites [2,3,4], supporting previous findings that modifications of histones H3 and H4 are critical for DSB repair [2,5]. CBP and p300 are required for the recruitment to DSB sites of KU70/ 80, which are key proteins involved in non-homologous end joining (NHEJ), a major DSB repair system [6]. Together, these studies suggest that histone acetylation by the CBP and p300 HATs facilitates chromatin remodeling at DSB sites, and that the resulting ‘‘relaxed’’ chromatin permits the recruitment and accumulation of DNA repair proteins at these sites

Methods

Results

Conclusion