Explorer A portable BRCA 1-HAC ( human artificial chromosome ) module for analysis of BRCA 1 tumor suppressor function

BRCA1 is involved in many disparate cellular functions, including DNA damage repair, cell-cycle checkpoint activation, gene transcriptional regulation, DNA replication, centrosome function and others. The majority of evidence strongly favors the maintenance of genomic integrity as a principal tumor suppressor activity of BRCA1. At the same time some functional aspects of BRCA1 are not fully understood. Here, a HAC (human artificial chromosome) module with a regulated centromere was constructed for delivery and expression of the 90 kb genomic copy of the BRCA1 gene into BRCA1-deficient human cells. A battery of functional tests was carried out to demonstrate functionality of the exogenous BRCA1. In separate experiments, we investigated the role of BRCA1 in maintenance of heterochromatin integrity within a human functional kinetochore. We demonstrated that BRCA1 deficiency results in a specific activation of transcription of higher-order alphasatellite repeats (HORs) assembled into heterochromatin domains flanking the kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction and extended previous observations that BRCA1 is required to silence transcription in heterochromatin in specific genomic loci. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation. INTRODUCTION BRCA1 is a well-known tumor suppressor gene, germ line mutations in which predispose women to breast and ovarian cancers. Since the identification of the BRCA1 gene, there have been numerous studies aimed at characterizing the diverse repertoire of its biological functions. BRCA1 is involved in multiple cellular pathways, including DNA damage repair, chromatin remodeling, X-chromosome inactivation, centrosome duplication and cell-cycle regulation (1– 7). A recent study has suggested a role in the epigenetic regulation of an oncogenic microRNA (8). BRCA1 associates with constitutive pericentromeric heterochromatin in nuclei (1). Further insight into the role of BRCA1 in pericentromeric heterochromatin and a significant link to maintaining global heterochromatin integrity has been recently gained by Zhu et al. (9). They showed that loss of BRCA1 results in transcriptional de-repression of tandemly repeated satellite DNA in mice and human BRCA1-deficient cells. This impairment of constitutive heterochromatin may lead to de-repression of the normally silenced genes that are located at the tandemly repeated DNA regions, probably through the loss of ubiquitylation of histone H2A. These effects on heterochromatin silencing could potentially account for some aspects of BRCA1 tumor suppression function. In their experiments, the authors employed a lentivirus vector expressing a BRCA1 cDNA to complement BRCA1deficiency. Such an approach may not completely recapitulate the physiological expression of the BRCA1 gene for several reasons. These include the lack of a strong copy number control of the transgene, the lack of alternative splice-forms when rescuing function with a cDNA and the absence of the intronic regions of the gene, which may include regulatory elements, and which, when spliced, will increase the *To whom correspondence should be addressed. Tel: +1 301 496 7941; Fax: +1 301 48