RESUMO
Recent results in living cells have now established the existence of levels of chromatin folding above the 30 nm fiber within interphase chromosomes. We discuss the potential functional impact of this large-scale chromatin organization, including its possible role in regulating gene expression.
Assuntos
Cromatina/fisiologia , Animais , Cromatina/química , Cromatina/genética , Regulação da Expressão Gênica , Humanos , Interfase , Conformação MolecularRESUMO
The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator-containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5' mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3' region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions.
Assuntos
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/genética , Alelos , Animais , Proteína BRCA1/química , Neoplasias da Mama/genética , Células CHO , Células Cultivadas , Cromatina/genética , Cricetinae , Feminino , Humanos , Rim/citologia , Mamíferos , Dados de Sequência Molecular , Mutação/fisiologia , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Transcrição , Ativação Transcricional/fisiologia , Técnicas do Sistema de Duplo-Híbrido , LevedurasRESUMO
Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.
Assuntos
Óperon Lac/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Receptor alfa de Estrogênio , Estrogênios/farmacologia , Genes Reporter , Histona Acetiltransferases , Ligantes , Microscopia de Fluorescência , Coativador 1 de Receptor Nuclear , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
The respiratory epithelium undergoes morphological and functional changes following exposure to single oxygen. However, mechanisms by which singlet oxygen causes cellular injury are unclear. The present experiments were designed to investigate the possibility that singlet oxygen, a highly reactive species, diffuses into respiratory epithelial cells. Of the various methods for detection of singlet oxygen, the electron spin resonance (ESR) spectrometric technique was judged to be most compatible and sensitive for use with cell suspensions. ESR spectrometry was used to monitor the singlet oxygen-mediated conversion of 2-(9,10-dimethoxyanthracenyl)-tert-butylhydroxylamine, (I), to 2-(9,10-dimethoxyanthracenyl)-tert-butylnitroxide, (II), and its corresponding endoperoxide, (III), in human bronchial epithelial cells treated with extracellularly generated singlet oxygen. In a second series of experiments, bronchial epithelial cells labeled with (I) were treated with singlet oxygen in the presence of 1,4-diazabicyclo[2.2.2]octane, a singlet oxygen quenching agent. The addition of this quenching agent eliminated the ESR spectrum corresponding with (II) and (III). This result is consistent with the quenching of singlet oxygen by 1.4-diazabicyclo[2.2.2]octane. Collectively, our results indicate that extracellularly generated singlet oxygen diffuses into human bronchial epithelial cells and that this process is a potentially important step in the cytotoxic action of singlet oxygen to the respiratory epithelium.
Assuntos
Brônquios/metabolismo , Oxigênio/metabolismo , Antracenos , Células Cultivadas , Deutério , Difusão , Espectroscopia de Ressonância de Spin Eletrônica , Epitélio/metabolismo , Radicais Livres , Humanos , Oxirredução , Marcadores de Spin , Superóxidos/metabolismoRESUMO
The purpose of this study was to determine if significant arsenic exposure was occurring at a Superfund site with elevated surface soil arsenic concentrations. A second objective was to determine the statistical relationship between the various methods of measuring arsenic exposure in humans. Random urine, 24-hr urine, hair, and fingernail samples were collected at the end of the workweek from 40 employees at an active pesticide manufacturing facility which had formerly produced arsenical pesticides. There was no indication of adverse health effects among the employees attributable to arsenic exposure. Mean urinary, hair, and fingernail concentrations of arsenic were well within normal values and indicated that significant arsenic exposure was not occurring among the employees. Random and 24-hr urine measurements were significantly correlated. Hair and fingernail results also were significantly correlated. Urine results did not correlate well with hair or fingernail results. Results of this study suggest that although there may be some individual variation, random and 24-hr urine arsenic results are not substantially different. For the purpose of screening for arsenic exposure, random urine samples may be an adequate and preferable test for those populations in equilibrium with their environment.