The Mechanism Of Hsf4b Transcription Activity Is Regulated By BAT1in Mouse Lens Epithelial Cells

BackgroundCongenital cataract is the main cause of blindness in children, about a third of of congenital cataract ishereditary, which autosomal dominant accounts for a high proportion. Inherited congenital cataract weremostly caused by gene mutations. the main pathological changes of congenital cataract are thedevelopmental disorders of the lens, fiber plate formation in lens, and eventually lead to the lens opaque.Reaserch have shown that the occurrence of congenital cataract is associated with the mutation of multiplegenes, such as the lens cell structure related gene mutation (alpha, bete-and gamma-crystallins and GJA3/A8, AQPO,BFSP2) and regulation transcription factor of lens development related genes mutations(PITX3PAX6, FOXE3, EYA1, MAF and Hsf4).Heat shock transcription factor4(Hsf4) plays a key role in children lens development. Hsf4has twoisoforms by the different splicing: Hsf4a and Hsf4b. Hsf4a acts as an inhibitor of the constitutiveexpression of heat shock genes. In contrast, Hsf4b acts as a transcriptional activator. Hsf4b is a form ofactivity in lens, it is mainly involved in regulation of small molecules heat shock protein (hsp25、γ-crystallin、α-crystallin) and fiber skeleton protein (imentin, filesin) protein expression. Study found thatloss of Hsf4b gene leads to the lack of downstream protein expression, such as Hsp25, gamma of crystallinF, gamma-S crystal protein, etc.These findings suggest that Hsf4b is an important transcription factor in anearly development of the lens. Hsf4b is regulated by phosphorylation, acetylation, ubiquitination, etc.However, regulating Hsf4b transcription activity of signaling pathways and Hsf4b regulated molecularmechanism in the early development of len are still unclear. Using yeast two hybrid experiments we have discovered a new and interacted with Hsf4protein-BAT1(also called UAP56,56kd, U2AF56-associated protein). BAT1protein belongs to DAED-boxfamily members, BAT1genes firstly cloned from pigs, it contains nine highly conservative structuredomain. It widely exists in many species, from bacteria to mammals, it is an ATP dependent RNA helicasewith ATPase hydrolytic activity. It involved in various metabolic processes such as RNA transcription、pre-mRNA splicing、 ribosomes and spliceosome assembly、 mRNA transport from the nucleus to thecytoplasm、 protein translation、 and maintain the stability of the mRNA and mRNA degradation and otherimportant life. This study analysis BAT1interacts with Hsf4b and colocalizates in the mice len epithelialcells. exploreing BAT1effects on Hsf4b regulatory protein, enriching the molecular mechanism ofcongenital cataract, for the early diagnosis and treatment of congenital cataract to provide theoretical basis.ObjectiveTo study len epithelial cells in mice BAT1gene interacts with Hsf4b and regulates mechanism ofHsf4b downstream protein expression.Method(1)Using pwzl-HA, pwzl-HA-Hsf4b, pbabe-HA-BAT1, pwzl-HA-Hsf4b+pbabe-HA-BAT1retrovirus supernatant infected hsf4-/-mice epithelial cells (mLEC/hsf4-/-), using blasticidin andpuromycin selection successful establish stable cell strains. Using BAT1K95E, Hsf4b+BAT1K95Eretrovirus supernatant infected hsf4-/-mice epithelial cells (mLEC/hsf4-/-), with puromycin selectionsuccessful establish stable cell strains.(2)Validating Hsf4b interacte with BAT1and intracellular localization by cell immunofluorescenceexperiments.(3) Validating BAT1effects on Hsf4b downstream genes in mRNA level by Realtime-PCR method, using western blot (western blotting) test method, validates that BAT1effects on Hsf4b downstream genesαB、hsp25in the expression of protein levels(4)Detecting BAT1regulates Hsf4b downstream gene αB-crystal protein gene promoter by Luciferaseassay.(5)Through the nuclear cytoplasm separation, Realtime-PCR method validates that BAT1effects on Hsf4b the downstream protein nulear output and Pull Down tests whether Hsf4b promotes BAT1ubiquitination.(6)Scince in the mRNA level BAT1gene has nuclear output function, and its95loci plays animportant role in nuclear output function.establishing pbabe-HA-BAT1K95E mutation plasmid,enzyme cutting、sequencing and identification mutations loci.(7)Validating mutation BAT1effects on Hsf4b downstream genes αB in mRNA level byRealtime-PCR method.Result(1)Len epithelial cells in mice(mLEC), BAT1interacts with hsf4b in the cell nucleus and positioning,at the same time BAT1inhibits αB promoter transcription activity of hsf4b downstream proteingene.BAT1also inhibits αB, hsp25expression of hsf4b downstream protein.(2)According to the biological function of BAT1, BAT1does not regulate gene αΒ nuclear output ofhsf4b downstream gene, at the same time BAT1inhibits downstream protein expression of hsf4b is not dueto BAT1nuclear output.At the same time, hsf4b cannot promote BAT1ubiquitination.ConclusionBAT1combines Hsf4b in vitro, at the same time, total positioning within the len epithelial cell nucleusin mice.BAT1reduces Hsf4b downstream gene αB, hsp25mRNA level, decreases αB, hsp25protein expression, and the effect has no obvious relation with comining capacity of BAT1.