RESUMO
The list of factors that participate in the DNA damage response to maintain genomic stability has expanded significantly to include a role for proteins involved in RNA processing. Here, we provide evidence that the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS) is a novel component of the DNA damage response. We demonstrate that FUS is rapidly recruited to sites of laser-induced DNA double-strand breaks (DSBs) in a manner that requires poly(ADP-ribose) (PAR) polymerase activity, but is independent of ataxia-telangiectasia mutated kinase function. FUS recruitment is mediated by the arginine/glycine-rich domains, which interact directly with PAR. In addition, we identify a role for the prion-like domain in promoting accumulation of FUS at sites of DNA damage. Finally, depletion of FUS diminished DSB repair through both homologous recombination and nonhomologous end-joining, implicating FUS as an upstream participant in both pathways. These results identify FUS as a new factor in the immediate response to DSBs that functions downstream of PAR polymerase to preserve genomic integrity.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Instabilidade Genômica/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Humanos , Lasers/efeitos adversos , Poli Adenosina Difosfato Ribose/genética , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Estrutura Terciária de Proteína , Proteína FUS de Ligação a RNA/genéticaRESUMO
Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.
Assuntos
Regulação da Expressão Gênica/imunologia , Histonas/metabolismo , Imunidade Inata/imunologia , Interferon gama/metabolismo , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Acetilação , Imunoprecipitação da Cromatina , Primers do DNA/genética , Citometria de Fluxo , Humanos , Interleucina-12/metabolismo , Interleucina-18/metabolismo , Microscopia Confocal , Células T Matadoras Naturais/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT4/metabolismo , Imagem com Lapso de TempoRESUMO
CRHSP-28 is a member of the tumor protein D52 protein family that was recently shown to regulate Ca(2+)-stimulated secretory activity in streptolysin-O-permeabilized acinar cells (Thomas, D. H., Taft, W. B., Kaspar, K. M., and Groblewski, G. E. (2001) J. Biol. Chem. 276, 28866-28872). In the present study, the Ca(2+)-sensitive phospholipid-binding protein annexin VI was purified from rat pancreas as a CRHSP-28-binding protein. The interaction between CRHSP-28 and annexin VI was demonstrated by coimmunoprecipitation and gel-overlay assays and was shown to require low micromolar levels of free Ca(2+), indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca(2+)-dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.