Genome-wide location analysis of BRCA1 in mammalian cells.

RUSTICI G; DE SIERVI A; CUI W; HAGGERTY CM; GARDNER K

Steamboat Springs, Co. USA.

Simposio; Systems Biology Keystone Symposium; 2007

Germline mutations of the breast cancer associated gene 1 (BRCA1) confer an increased risk for breast and ovarian cancers. Several functions have been attributed to BRCA1 including regulation of cell cycle progression, DNA damage signaling and repair, maintenance of genomic integrity and regulation of various transcriptional pathways. Although several BRCA1 interacting proteins and target genes have been identified, the molecular mechanisms by which it carries out these functions are not fully understood. The aim of this study is to profile the transcriptional targets of BRCA1 by global genome location analysis and describe the distribution of BRCA1 across the genome before and after genotoxic stress. Exponentially growing Jurkat cells untreated or exposed to UV irradiation are formalin crosslinked and chromatin immunoprecipitation is carried out using an affinity purified BRCA1-specific antibody, raised against exon 11.  The BRCA1-bound DNA is then hybridized to Nimblegen high-density tiling oligo arrays.  A “probe-centered” algorithm is used to calculate a p-value of BRCA1 enrichment for each promoter region. Over 1000 genes are enriched for BRCA1 in untreated cells (list A) and 567 following UV irradiation (list B), showing a clear redistribution of BRCA1 after genotoxic stress. In list A, Gene Ontology (GO) associations are enriched for the terms ´Chromatin assembly´, ´DNA damage response´ and ´Regulation of cell cycle´. In list B, the GO terms ´M phase´ and ´Mitotic checkpoint´ are over-represented, suggesting a specific role for BRCA1 in controlling mitotic events following genotoxic stress. Preliminary results show significant overlap between the potential BRCA1 targets identified in this location study and the genes with altered gene expression following BRCA1 knockdown by RNA interference, in human prostate and breast cancer cells. Expression of selected BRCA1 targets is also altered in mouse embryonic fibroblasts carrying mutation of Brca1 exon 11 (Brca1D11/D11). These findings suggest that a functional BRCA1 protein is required for transcriptional regulation of several genes involved in mitotic progression and chromosome maintenance. Results of this study define direct transcriptional targets of BRCA1 in human cells and provide a foundation for elucidating the transcriptional network through which BRCA1 maintains genome integrity under normal and stress induced environmental conditions.