Abstract
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 (Brg1), the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brg1 interact with Signal transducer and activator of transcription (Stat) 1 in vitro. However, Stat1 in its native form only recruits hBrm to IFNγ-activated sequences (GAS) of individual genes; by contrast, in a stress-induced phosphorylated form, Stat1 mainly binds to Brg1. Under basal conditions, hBrm is recruited by native Stat1 to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90α 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 Stat1 is increased, and binds and recruits Brg1 to the GAS, leading to elevated induction of the gene. This hBrm/Brg1 switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; however, this switch only occurs in specific cell types upon exposure to IFNγ. Regardless of the stimulus, the hBrm/Brg1 switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brg1 switch is an indicator of the responsiveness of a gene to heat-shock or IFNγ stimulation and may represent an “on-off switch” of gene expression in vivo.
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Acknowledgements
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).
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Supplementary information
Supplementary information, Figure S1
Efficacy of the hBrm and Brg1 knockdown with their respective siRNA. (PDF 87 kb)
Supplementary information, Figure S2
The interaction of Stat1 with Brg1 or with hBrm in the nuclear extracts from Jurkat cells was not sensitive to DNase I digestion (1u/100μg) at room temperature for 10min. (PDF 199 kb)
Supplementary information, Figure S3
Construction of K1541R/K1543R and K1534R/K1535R plasmids of hBrm. (PDF 75 kb)
Supplementary information, Figure S4
Efficacy of Stat1 knock down by specific shRNA. (PDF 99 kb)
Supplementary information, Figure S5
Effects of heat shock and IFNγ on the expression of IRF-1 gene and a Stat1 mediated hBrm to Brg1 switch at the GAS region of IRF-1 gene in Jurkat. (PDF 52 kb)
Supplementary information, Figure S6
p300 recruiting to the GAS under heat shock in Jurkat and Raji cells. (PDF 42 kb)
Supplementary information, Table S1
The oligonucleotides used in Brg1 and Brm RNAi constructs (PDF 6 kb)
Supplementary information, Table S2
Primers used for the construction of truncated Stat1 (PDF 9 kb)
Supplementary information, Table S3
Primers used in K/R mutations of hBrm-R, Bm-uR & Bm-dR (PDF 10 kb)
Supplementary information, Table S4
Primers in ChIP assays. (PDF 11 kb)
Supplementary information, Table S5
Primers for mRNA expression of the genes with qRT-PCR (PDF 11 kb)
Supplementary information, Table S6
qPCR Primers used in restriction enzyme accessibility assay (PDF 25 kb)
Supplementary information, Table S7
The primers used for construction of mutated mSin3a. (PDF 9 kb)
Supplementary information, Data S1
Supplementary Materials and Methods (PDF 81 kb)
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Zhang, Y., Cheng, Mb., Zhang, Yj. et al. A switch from hBrm to Brg1 at IFNγ-activated sequences mediates the activation of human genes. Cell Res 20, 1345–1360 (2010). https://doi.org/10.1038/cr.2010.155
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DOI: https://doi.org/10.1038/cr.2010.155
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