Regulatory Effect Of Lysine Mutation On Hsf4b Transcriptional Activity

BackgroundCongenital cataract is a kind of eye disease, which occurs in the children and causes around22-30%ofchildren blindness. Its main clinical symptom is lens opacity. The abnormal genetic mutations are thedominant pathological causes.Hsf4has been found to be a dominant regulator of postnatal lens development clinically and in theanimal model. The missense mutation in its DNA binding domain or mutation in splicing junctions hasbeen closely related to the human family inheritage chromosom autosomal dominant or recessive cataracts.Knock down Hsf4gene in mouse tissues, which can increase lens epithelial cell proliferation and abnormalfiber cell differentiation, can induce the cataract formation at postnatal age.Hsf4has two splicing forms Hsf4a and Hsf4b, Hsf4b is the unique that expressed in postnatal lensepithelial and fiber cells. It can upregulate the expression of small heat shock proteins (Hsp25, alphaB-crystallin, r-crystallin) and the skeletal proteins (VP49, vimentin and skap2) and down-regulate theexpression of FGF1and7in lens tissues. However, the signal pathway that regulates Hsf4transcriptionactivity in postnatal age is still unclear. Hsf4b transcriptional activity is regulated by the posttranslationalphoshphorylation and sumoylation. It is reported that phosphorylation of S298and sumoylation of K293can inhibit Hsf4b transcription activity. Our previous data indicate that phosphorylation of S298canenhance the interaction between Hsf4b and Daxx2, which leads to downregulation of Hsf4b transcriptionactivity. In addition, whether Hsf4b is regulated by other posttranslational modification (e.g. acetylationand ubiqutination) is still unknown. Lysine is the dominant amino acid that is used to be modified bysumoylation, acetylation and ubiquitination. There are numbers of lysine in Hsf4b protein sequence.Whether these lysines are involved in the regulation of Hsf4b transcription activity will be examed in thisproject. With the GPS software we identify K64, K206, K224, K287, K293, K336and K434are potentialto be modified by either sumoylation or acetylation. With site-mutation we find that mutation of K64G,K206G and K224G have inhibitory effects on Hsf4b while mutation of K287G, K293G, K336G and K434Gcan upregulate Hsf4b transcription activity, the signal pathways that controls these lysine modification is still under investigation.AimsWith the site-mutation method we will determine the regulatory effects of lysines on Hsf4btranscription activity and explore their signal pathways underlying Hsf4b during lens development. Ourstudy will be significant for the discovery of the molecular mechanism of Hsf4b-feficency-induced cataract,early provention and diagnosis of Hsf4b-related congenital cataracts.Methods1Design and synthesis seven pair primers which can change the Hsf4b cDNA sequences of192bp、618bp、672bp、861bp、879bp、1008bp and1302bp lysine coden AAG/AAA into glycine coden GGC.2Quicker-Site-mutation PCR is used to mutation of K64G, K206G, K224G, K287G, K293G, K336G andK434G by using plasmid pWZL-blast-HA-Hsf4b as templete, and generate seven constructs.3Reconstitution of wild-Hsf4b and lysine-mutant of Hsf4b into the mouse Hsf4b-/-lens epithelial cells byretrovirus-mediated infection. The plasmids of pWZL-blast-HA-Hsf4b and pWZL-blast-HA-Hsf4bmutants were transfected into the retoviral packaging cell line293phoenix cells respectively. After48houres, the supernatants, which contain the virus, were applied to infect the mLEC/Hsf4-/-cellsrespectively and generated the stable cell lines.4Western blotting assay is used for detecting the protein expression of Hsf4b and Hsf4b and theirdownstream targets of Hsp25and αB-crystallin.5The luciferase assay is used to determine the regulatory effects of Hsf4b and Hsf4b mutants on thepromoter activity of αB–crystalline gene.Results1Sucessfully mutating the seven lysines into the glycine and generated the plasmids.2Establishing the stables lens epithelial cell lines that express sham, wild-type Hsf4b and Hsf4b mutantsrespectively.3Western blotting results show that the expression level of Hsf4b mutants is almost as same as thewild-type Hsf4b.The expression of downstream proteins Hsp25and αB-crystallin were down-regulatedin mLEC/HA-Hsf4b/K64G, mLEC/HA-Hsf4b／K206G and mLEC/HA-Hsf4b／K224G, but wereupregulated in the cell lines of mLEC/HA-Hsf4b／K287G, mLEC/HA-Hsf4b/K293G, mLEC／ HA-Hsf4b/K336G and mLEC/HA-Hsf4b/K434G comparing to their expression in the cell line ofmLEC/HA-Hsf4b.4The result of luciferase assay showed that B-crystallin promoter activity, which is upregulated in themLEC/HA-Hsf4b cells compared to the mLEC/mock, is downregulated in the cell lines ofmLEC/HA-Hsf4b/K64G but increased in the cell line of mLEC/HA-Hsf4b/K293G compared to theiractivity in mLEC/HA-Hsf4b cell line.ConclusionsThe Hsf4b transcription activity is regulated by the potental posttranslational modification of lysine.The K64, K206and K224have the active regulatory roles on Hsf4b transcription activity while K287,K293, K336and K434have downregulation of Hsf4b transcription activity.