The Role Of BAT1Gene On Regulate The Heat Shock Factor Hsf4b

BackgroundCataract, or opacification of all or part of the lens of the eye, reduces optical performance, most commonly manifested by decreased visual acuity, glare and decreasedcontrast sensitivity. It can be divided into either the early-onset (congenital) type or the Inherited type. Congenital cataract is particularly serious because it has the potential for inhibiting visual development, resulting in permanent blindness.The knowledge of the genetic background has increased considerably during the past decennia, mainly based on linkage strategies in large families. About more than40cataract-associated loci are known, of which25represent identified genes, and the number of mutations exceeds more than100.Mutations causing developmental cataracts mainly involve proteins with structural and chaperone functions, including α(?)-,β(?)-, and γ-crystallins. Another group includes the lens-specific transmembrane gap junction protein genes GJA3and GJA8, and the membrane protein genes MIP and LIM2. A third group of genes represents the lens-associated transcription factors HSF4, PITX3, MAF, PAX6, and FOXE3.In humans, missense mutation in the hsf4gene causes cataract, and mice bearing a targeted disruption of the hsf4gene exhibit defects in lens fiber cell differentiation and early cataract formation.HSF4belongs to the family of heat shock transcription factors (HSFs) that can bind to the heat shock element (HSE) and activate the transcription of downstream targetgenes including heat shock proteins (Hsp70, Hsp90,Hsp27, Hsp82) in response to different stresses。 The heat shock factor Hsf4possesses alternative splice variants leading to generation of two isoforms: Hsf4a that has transcriptional repressor activity and Hsf4b that has transcriptional activator properties.The recently results suggest that Hsf4b plays a critical role in the lens fiber cell maturation.While the developmental role of Hsf4is becoming clear, themode of Hsf4regulation of transcription is not yet understood.To examine the molecular mechanisms controlling the transcriptional activity of Hsf4b, we used Hsf4b as a bait in a yeast two-hybrid screening system and have identified a novel protein called BAT1(56kDa U2AF65-associated protein)that interacts with Hsf4b.BAT1is a member of DEAD box family of ATP-dependent RNA helicases. BAT1has both RNA-stimulated ATP binding/hydrolysis activity and ATP-dependent RNA unwinding activity. Studies in different organisms indicate that BAT1protein is not only essential in pre-mRNA splicing but is also important in mRNA nuclear export and cytoplasmic mRNA localization.ObjectivesIn this study, by constructing a several of BAT1expression vector to to verify the interaction between BAT1and hsf4b, and find its site of action, as well as how BAT1regulates the downstream gene of hsf4b.Methods1.Using Polymerase chain reaction (PCR), obtain the BAT1the cDNA sequence, BAT1PCR products were digested with Bamh land Not I, then cloned into the eukaryotic expression vector pEBG pcDNA3.0to got pEBG-BAT1pcDNA3.0-T7-BAT1plasmid. BAT1PCR products were digested with Bamh I and Sal I, then cloned into the prokaryotic expression vector pGEX-4T3-4T3to got the pGEX-4T3-BAT1plasmid.2The authenticity of the above plasmid was confirmed by sequencing and then pEBG-BAT1pcDNA3.0-T7-BAT1plasmid transfected into293T cells For eukaryotic expression。identificated the BAT1protein by using SDS-PAGE and Western blot experiments。pGEX-4T3-BAT1plasmids were transformed into BL21competent cells induced the expression of BAT1protein, identificated the vitro expressed BAT1protein by using SDS-PAGE。3To verify the interactions between BAT1protein and hsf4b protein by using vivo and vitro pull-down assay, as well as through the pull-down assay to verify BAT1interact with which functional areas of hsf4b.4To test the effection on how BAT1regulate the expression of downstream gene alpha B-crystallin protein (alpha-Crystallin B) of hsf4bResults.1. The eukaryotic expression vector pEBG BAT1,pcDNA3.0-T7-BAT1, the prokaryotic vector pGEX-4T3-BATlwas built and we obtain the corresponding expression of BAT1protein.2. The in vivo pull-down assay experimental verifed the interaction between BAT1and hsf4b; in vitro pull-down assay also verified the BAT1and hsf4b interaction. And preliminary proved BAT1interact with hsf4b at the C-terminal amino acid in hsf4b.3. The luciferase assay preliminary proved the inhibition of BAT1on hsf4b downstream gene alpha B-crystallin protein (alpha-Crystallin B).Conclusions1. BAT1and hsf4b interact with each other both in vivo and in vitro2. BAT1interact with hsf4b at the C-terminal amino acid in hsf4b.3. BAT1inhibited the expression of hsf4b downstream gene alpha B-crystallin protein (alpha-Crystallin B).