Identification Of The Interaction Of HSF4and SNF8

Congenital cataract is one of the main cause for childhood blindness, with anincidence of about0.3-1.5/1000, nearly half of the congenital cataracts have beenassociated with inheritance. So far,26causative genes and40genetic loci have beenidentified which mutated may lead to congenital cataracts. Heat shock transcription factor4(HSF4) plays a key role in the regulation of the early development of the lens as atranscription factor. Missense-mutations in the HSF4DNA binding region have beenreported to cause chromosome dominant cataracts. HSF4has two isforms, HSF4a andHSF4b. In the lens of mouse, HSF4b positively regulate the heat shock protein expressionbut negatively regulate the expression of the FGF subfamily members. However, thespecific molecular mechanism of HSF4transcriptional activity is unclear. Thus to find theprotein interaction network between HSF4and other len’s proteins is key to understandHSF4function, and it’s crucial in lens development and the molecular pathologicalmechanism for cataract.In the previous study, we have found that HSF4may interact with SNF8throughyeast two-hybrid. In this thesis, we used GST pull-down and co-immunoprecipitation toconfirm the interaction between HSF4and SNF8.We performed immunohistochemical assay and found that SNF8expressed in themouse lens. Western blot showed SNF8is present in the human lens epithelial (HLE) cells.After cloning the full-length SNF8gene from HLE cell cDNA, constructed SNF8expression vectors by subcloning SNF8into3×Flag-cmv7.1-vector and transfected toHLE cells. The pGEX-6P-1-Hsf4b plasmid was transformed in Escharichia coli strainBL21, with IPTG induction, incubated with the cell Lysates containing SNF8protein afterthe purifion of glutathioneTM4B agarose beads. And finally, the polyacrylamide gelelectrophoresis analysis showed that HSF4b interacted with SNF8.We further validate the interaction between HSF4b and SNF8by usingcoimmunoprecipitation. p3×FLAG-CMV-7.1-SNF8and pEGFP-Hsf4b-wt plasmids wereco-transfected into HLE cells. After48hours, Cells were lysed and the anti-tag antibodyprotein was added to the cell lysate, and then incubated with Protein A/G for a period of time. The denaturing polyacrylamide gel electrophoresis showed that HSF4b interactedwith SNF8. To reveal the key functional elements of HSF4, through series missingmethods, we verify the different domains of the HSF4b to affect the interaction process.We found that the DBD domain and HR A/B domain of HSF4b played a crucial role in theinteraction between HSF4b and SNF8, but not DHR domain.