Analysis of the Bim/Dlc protein complex regulating the survival of hematopoietic cells under the control of cytokines.

点击翻译按钮获取中文摘要

Expression control systems were analyzed by DNase hypersensitivity assay, reporter analysis, gelshift assay, nuclear run off assay, and experiments monitoring the half life of mRNA. By reporter assay, we isolated cis-elements, which upregulate(several hundreds base pairs upstream of the transcriptional initiation site) or downregulate (in the first intron) the transcriptional efficiencies. Unexpectedly, the silencer sequence in the first intron is cytokine independent. Moreover, nuclear run off assay suggested that the expression of Bim is not controlled by transcriptional mechanisms, but by the regulation of the half-life of its message. Therefore, Bim mRNA is likely regulated by modification of mRNA degradation. In vitro assay, the half-life of Bim mRNA was elongated by cytokines. We found that this is mediated by heat shock cognate protein(Hsc70), which binds and stabilizes the mRNA. The binding potential of Hsc70 to mRNA is turned out to be regulated by cochaperons such as Hsp40, Hip, and Bag proteins.