title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function.
abstract: 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 alpha-satellite 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.
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Artem V
authors.initials: AV
authors.lastname: Kononenko
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Bansal
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Nicholas C O
authors.initials: NC
authors.lastname: Lee
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN 46202, USA.
authors.forename: Brenda R
authors.initials: BR
authors.lastname: Grimes
authors.affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA, Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA kouprinn@mail.nih.gov.
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
mesh_terms: D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism* / Q000502:physiology; D016466:CHO Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D022222:Chromosomes, Artificial, Human; D006224:Cricetinae; D003412:Cricetulus; D019398:Genes, BRCA1; D006570:Heterochromatin / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D013552:Swine
chemical_list: D019313:BRCA1 Protein; D006570:Heterochromatin