The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma （hBrm） and Brahma-relate...The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma （hBrm） and Brahma-related gene 1 （Brgl）, the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brgl interact with Signal transducer and activator of transcription （Stat） 1 in vitro. However, Statl in its native form only recruits hBrm to IFNy-activated sequences （GAS） of individual genes; by contrast, in a stress- induced phosphorylated form, Statl mainly binds to Brgl. Under basal conditions, hBrm is recruited by native Statl to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90a gene, which shows a compact chromatin structure. Upon heat-shock, hBrm is acetylated by p300 and dissociates from the co-repressor complex, which the phosphorylated St^tl is increased, and binds and recruits Brgl to the GAS, leading to elevated induction of the gene. This hBrm/Brgl switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; how- ever, this switch only occurs in specific cell types upon exposure to IFNy. Regardless of the stimulus, the hBrm/Brgl switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brgl switch is an indicator of the responsiveness of a gene to heat-shock or IFNy stimulation and may represent an ＂on-off switch＂ of gene expression in vivo.展开更多
AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were o...AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.展开更多
基金Acknowledgments We thank Drs XY Fu, CM Horvath, AN Imbalzano, HB Zhang, S Cadelwood and K Shuai for kindly providing plasmids, antibodies and chemicals used in this work. We thank Dr Robert A Casero, Jr of the Johns Hopkins University School of Medicine for his critical reading of the manuscript, and Dr Weimin Zhong of the Yale University for his contribution to the work. This work was supported by the National Natural Science Foundation of China （90408007, 30871382 and 30721063）, the National Basic Research Program of China （973 Program） （2005CB522405）, the National Key Scientific Program （2011CB964902） and Special Funds of State Key Laboratories （2060204）.
文摘The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma （hBrm） and Brahma-related gene 1 （Brgl）, the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brgl interact with Signal transducer and activator of transcription （Stat） 1 in vitro. However, Statl in its native form only recruits hBrm to IFNy-activated sequences （GAS） of individual genes; by contrast, in a stress- induced phosphorylated form, Statl mainly binds to Brgl. Under basal conditions, hBrm is recruited by native Statl to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90a gene, which shows a compact chromatin structure. Upon heat-shock, hBrm is acetylated by p300 and dissociates from the co-repressor complex, which the phosphorylated St^tl is increased, and binds and recruits Brgl to the GAS, leading to elevated induction of the gene. This hBrm/Brgl switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; how- ever, this switch only occurs in specific cell types upon exposure to IFNy. Regardless of the stimulus, the hBrm/Brgl switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brgl switch is an indicator of the responsiveness of a gene to heat-shock or IFNy stimulation and may represent an ＂on-off switch＂ of gene expression in vivo.
基金Supported by The Dutch Cancer Society(grant.UvA2002-2604)
文摘AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.
