RESUMO
An abnormal differentiation state is common in BRCA1-deficient mammary epithelial cells, but the underlying mechanism is unclear. Here, we report a convergence between DNA repair and normal, cultured human mammary epithelial (HME) cell differentiation. Surprisingly, depleting BRCA1 or FANCD2 (Fanconi anemia [FA] proteins) or BRG1, a mSWI/SNF subunit, caused HME cells to undergo spontaneous epithelial-to-mesenchymal transition (EMT) and aberrant differentiation. This also occurred when wild-type HMEs were exposed to chemicals that generate DNA interstrand crosslinks (repaired by FA proteins), but not in response to double-strand breaks. Suppressed expression of ΔNP63 also occurred in each of these settings, an effect that links DNA damage to the aberrant differentiation outcome. Taken together with somatic breast cancer genome data, these results point to a breakdown in a BRCA/FA-mSWI/SNF-ΔNP63-mediated DNA repair and differentiation maintenance process in mammary epithelial cells that may contribute to sporadic breast cancer development.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/prevenção & controle , Diferenciação Celular , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Células Epiteliais/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Glândulas Mamárias Humanas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Acetaldeído/farmacologia , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cisplatino/farmacologia , DNA Helicases/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Formaldeído/farmacologia , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Mutação , Proteínas Nucleares/genética , Fenótipo , Interferência de RNA , Transdução de Sinais , Fatores de Transcrição/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Endogenous BRCA1 p220 expression peaks in S and G2 when it is activated, and the protein participates in certain key DNA damage responses. In contrast, its expression is markedly reduced in G0/G1. While variations in transcription represent a significant part of p220 expression control, there is at least one other relevant process. We found that a microRNA, miR-545, that is expressed throughout the cell cycle down-modulates endogenous p220 mRNA and protein abundance directly in both G0/G1 and S/G2. When miR-545 function was inhibited by a specific antagomir, endogenous p220 expression increased in G0/G1, and aberrant p220-associated DNA damage responses and de novo DNA strand breaks accumulated. Analogous results were observed upon inhibition of miR-545 function in S/G2. Both sets of antagomir effects were mimicked by infecting cells with a p220 cDNA-encoding adenoviral vector. Thus, strand breaks were a product of p220 overexpression, and their prevention by miR-545 depends on its modulation of p220 expression. Breaks were also dependent on aberrant, overexpressed p220-driven recruitment of RAD51 to either spontaneously arising or mutagen-based DNA damage sites. Hence, when its level is not physiologically maintained, endogenous p220 aberrantly directs at least one DNA repair protein, RAD51, to damage sites, where their action contributes to the development of de novo DNA damage. Thus, like its loss, a surfeit of endogenous p220 function represents a threat to genome integrity.
Assuntos
Proteína BRCA1/genética , Ciclo Celular/fisiologia , Dano ao DNA/genética , Regulação da Expressão Gênica , Proteína BRCA1/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Reparo do DNA , Células HEK293 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Rad51 Recombinase/metabolismo , Raios UltravioletaRESUMO
Dicer is central to the RNA interference (RNAi) pathway, because it is required for processing of double-stranded RNA (dsRNA) precursors into small RNA effector molecules. In principle, any long dsRNA could serve as a substrate for Dicer. The X inactive specific transcript (Xist) is an untranslated RNA that is required for dosage compensation in mammals. It coats and silences 1 of the 2 X chromosomes in female cells and initiates a chromosomewide change in chromatin structure that includes the recruitment of Polycomb proteins, but it is largely unknown how Xist RNA mediates these processes. To investigate a potential link between the RNAi pathway and X inactivation, we generated and analyzed Dicer-deficient embryonic stem (ES) cells. In the absence of Dicer, coating by Xist RNA, initiation of silencing, and recruitment of Polycomb proteins occur normally. Dicer ablation had modest effects on the steady-state levels of spliced Xist RNA. Together our data indicate that the RNAi machinery is not essential for the initiation of X inactivation.
Assuntos
RNA Helicases DEAD-box/deficiência , Endorribonucleases/deficiência , Inativação do Cromossomo X/genética , Animais , Cruzamentos Genéticos , RNA Helicases DEAD-box/metabolismo , Doxiciclina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endorribonucleases/metabolismo , Feminino , Inativação Gênica/efeitos dos fármacos , Genes Ligados ao Cromossomo X , Endogamia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Genéticos , Proteínas do Grupo Polycomb , RNA Longo não Codificante , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Ribonuclease III , Tetraciclina/farmacologia , Cromossomo X/genética , Inativação do Cromossomo X/efeitos dos fármacosRESUMO
BRCA1, a breast and ovarian cancer-suppressor gene, exerts tumor-suppressing functions that appear to be associated, at least in part, with its DNA repair, checkpoint, and mitotic regulatory activities. Earlier work from our laboratory also suggested an ability of BRCA1 to communicate with the inactive X chromosome (Xi) in female somatic cells (Ganesan et al., 2002). Xiao et al. (2007) (this issue of Cell) have challenged this conclusion. Here we discuss recently published data from our laboratory and others and present new results that, together, provide further support for a role of BRCA1 in the regulation of XIST concentration on Xi in somatic cells.