The SP Family Transcription Factors Act As Major Regulators in the Transcription of the Major Heat Shock Protein, HSP90β in lens epithelial cells

Abstract

Purpose : Heat shock protein 90 (HSP90) plays fundamental role in protecting various biological systems from stress condition and also from pathogenesis. Previous studies have shown that HSF1 plays an important role in regulating expression of HSP90. Whether other transcription factors also play essential roles in regulating expression of HSP90 remains largely unknown. In the present study, we have demonstrated that except for HSF1, the SP family transcription factors including SP1/SP3/SP4 also play essential roles in regulating HSP90β transcription in lens epithelial cells.

Methods : Gel mobility shifting assays were used to determine binding of SP family transcription factors to various HSP90β gene promoter and enhancer regions. ChIP assays were used to confirm the binding of different SP factors to the HSP90β gene promoter and enhancer regions. Various reporter gene constructs driven by different promoter and enhancer regions of the HSP90β gene were generated and used to assay the relative strength of different HSP90β gene promoter and enhancer regions. CRISPR/Cas9 technology was used to create various mutants. QRT-PCR and Wes were used to analyze mRNA and protein expression levels.

Results : In the proximal promoter and downstream enhancer regions, numerous SP1/SP3/SP4 binding sites were identified. These cis-elements were able to mediate binding by SP family transcription factors via EMSA and ChIP, and most of these elements also provide positive regulation on the expression of HSP90β. The cis-elements in different promoter and enhancer regions have differential strength in mediating control of HSP90β expression.

Conclusions : The SP family transcription factors play an important role in regulating transcription of the major heat shock protein HSP90β in lens epithelial cells. Among different SP family transcription factors, SP1 is the most important factor in regulating HSP90β expression. Supported by National Natural Science Foundation of China (Grants #81970787, #82000876, #81770910) and Natural Science Foundation of Guangdong Province and Guangdong City Joint Program of China (Grant # 2019B1515120014)，and the Fundamental Funds, 3030901010110 of the State Key Laboratory of Ophthalmology of Zhongshan Ophthalmic Center.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.