The Study On The Mechanism Of The Expression Of Human Hsp90 Genes Controlled By A BTB/POZ Domain Protein GRP

The study of the expression and the control of gene on the chromatin level is one of hot spot at present. The two classes of complexes involved in chromatin remodelinghave been investigated more extensively. They are ATP-dependent complexes and chromatin-modifying complexes. Little is known about a third class of protein complexes that disrupt the normal nucleosome pattern by forming nonhistone chromatin structures. Drosophila GAF contains BTB/POZ domain. GBP, of that GAF is a component, is representative of this kind of nonhistone complexes. The complexes forming nonhistone chromatin structures regulate the expression of target gene containing GAGA element by chromatin remodeling.Fo observe if this kind of nonhistone complexes exists in human Jurkat cell, , we identified a GAGA element binding protein related protein, GRP using yeast one-hybrid system. By a competitive RT-PCR, this paper investigated mainly the effect of GRP on the promoter activity of its GAGA element-containing target gene ftz,hsp90αand hsp90βon the chromatin level.The promoter of Drosophila ftz gene is constructed into pREP4mCAf episomal vector capable of simulating chromatin structures. It is found that the CAT reporter gene driven by wild-type ftz promoter can express better than the CAT reporter gene driven by mutant-type ftz promoter in human Jurkat cell. The relative promoter activity of wild-type and mutant-type ftz gene is 1.5 (wild-type) and 0.5 (mutant-type) respectively ,suggesting that the factor similar to Drosophila GAF exists in human Jurkat cell.GRP enhances the promotion of GAF to the expression of CAT reporter gene driven by wild-type ftz gene promoter, but GRP does not about mutant-type ftz gene promoter. The results indicate that the complexes similar to GBP exist in human Jurkat cell.HsP90 is an important member of HSP protein family. HSP90 usually constitutively expressed in most mammalian cell types and can be further induced by heat shock and other stresses. HSP90 proteins are molecular chaperones that associated with cellar signal transduction, steroid hormone response, and cell cycle regulation, they are critical to the survival of cell in the normal and pathological conditions. So it is very meaningful for us to study the regulatory mechanisms of human hsp90 genes.We found a few of GAGA element in the hsp90αpromoter region ( -1756～+37 ) and hsp90βpromoter region ( -943～+1531 ) by searching them for the sites of transcriptional factor. Rc/CMV-GAF was transfected into human Jurkat cell with pREP4mCAT-hsp90α( -1756～+37 ) or pREP4mCAT- hsp90β( -943～+1531 ). When Rc/CMV-GAF is transfected at 37℃, GAF promotes the expression of CAT reporter gene driven by hsp90αpromoter in a dose-dependent manner. The effect is marked after heat shock for an hour at 42℃. Under the above experimental condition the change of CAT reporter gene driven by hsp90βpromoter is similar to that of hsp90α. Rc/CMV-GAF and pCDNA6-GRP was transfected into human Jurkat cell with pREP4mCAT-hsp90αor pREP4mCAT-hsp90β. GRP and GAF cooperatively promote the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. The mechanism regulating hsp90αor hsp90βpromoter by GRP and GAF may be similar to that regulating ftz gene promoter by GRP and GAF.Sodium butyrate is a common histone deacetylase inhibitor. The sense- or antisense- expressional plasmids of GRP were cotransfected into human Jurkat cell with pREP4mCAT-hsp90αor pREP4mCAT-hsp90β. 36 hours later the cells were treated with 2mM sodium butyrate for 12 hours. The results show that the 2mM sodium butyrate markedly enhances the promotion of GRP to the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. We assume that the promotion of GRP to hsp90αor hsp90βpromoter may involve histone deacetylation by Sodium butyrate. p300 is an irnportant histone acetyltransferase, p300 or p300△h and GRP were transfected into Jurkat cells, p300 and GRP synergistically promote the expression of CAT reporter gene driven by hsp90βpromoter, p300 and GRP do not about hsp90α, p300△h (histone acetyltransferase domain is deleted) can not affect the promotion of GRP to the expression of CAT reporter gene driven by hsp90αor hsp90βpromoter. These results suggest that p300 may take part in the regulation of GRP to hsp90αor hsp90βgene by histone acetyltransferase domain of it.GRP can regulate the distribtion of cell cycle. When the Jurkat cells were transfected with GRP, the percentage of G1-phase cells increases and the percentage of S-phase cells decreases, suggesting that GRP may blocks the G1-to-S transition. The overexpression of GRP can lead to cell apoptosis.Taken together, we get the following conclusion: The factor similar to Drosophila GAF or complexes similar to GBP may exist in human Jurkat cell. The mechanism regulating hsp90αor hsp90βpromoter by GRP and GAF may be similar to that regulating ftz gene promoter by GRP and GAF. The promotion of GRP to hsp90αor hsp90βpromoter may involve histone deacetylation by Sodium butyrate. P300 may take part in the regulation of GRP to hsp90αor hsp90βgene by histone acetyltransferase domain of it. GRP can regulate the distribtion of cell cycle. The overexpression of GRP can lead to cell apoptosis.