Regulation of alphaB-crystallin promoter by the BAF complex.

摘要

The BAF complex regulates transcription by modifying or altering chromatin structure. BAF complex is not known to bind DNA in a sequence specific manner, yet it recognizes specific genes. Protein factors such as activators and co-activator may assist in the targeting of the BAF complex. DNA microarray analyses of genome-wide gene expression profiles showed that the alphaB-Crystallin gene was one of 80 genes up-regulated by transient expression of BRG1, in SW-13 cells. Therefore, the alphaB-Crystallin promoter can serve as an ideal model or gene to elucidate the mechanism of how the BAF complex is targeted to the promoter of specific genes.; The alphaB-Crystallin promoter was first characterized by systematic deletion and mutation analyses in SW-13 cells, which lack the essential ATPase subunit of the BAF complex, BRG1. Co-transfection of the promoter reporter constructs with a BRG1 expression construct defined the promoter region that mediates the activity of the BAF complex. A thirty base pair region between -90 to 60 relative to the transcription start site was identified as the DNA element that mediates the activation of the alphaB-Crystallin promoter by BRG1. This region was shown to be packaged into a positioned nucleosome as indicated by Micrococcal Nuclease and Ligation Mediated-Polymerase Chain Reaction assays. Gel shift and competitive binding analyses demonstrated that the DNA element contained an AT-rich sequence that was bound by a sequence specific-DNA binding protein, and antibody supershift confirmed that the bound protein was HMG-I/Y protein. The functional relationship between HMG-I/Y and the BAF complex was ascertained via Western blot, RT-PCR and siRNA-HMG-I/Y procedures. These assays demonstrated that the lack of the HMG-I/Y protein significantly affected the inducibility of the BGR1 in the expression of alphaB-Crystallin gene.; Although we have not determined that HMG-I/Y protein interacts with the BAF complex, it cooperates with the BAF complex to regulate the expression of alphaB-Crystallin gene.