Abstract
Purpose :
CREB is a transcription factor and its most prominent function is to control the synaptic plasticity associated with long-term memory. In our recent studies, we demonstrated that CREB regulates oxidative stress-induced apoptosis and aging in lens by inhibiting αB-crystallin expression but promoting P300-P53-Bak/Bax signaling axis (Wang L et al. 2021. Aging Cell). Whether CREB is capable to regulate lens differentiation remains largely unknown. Here, in the present study, we demonstrate that CREB directly regulates Pax6 and other genes to control differentiation of mouse lens.
Methods :
CRISPR/Cas9 Technology was used to create mouse model CREB-S133A. RNAseq analysis was used to compare the transcriptional activity of wild type and mutant CREB-S133A.Gel mobility shifting assays were used to determine binding of CREB to Pax6 gene promoter. ChIP assays were used to confirm the binding of CREB to the Pax6 gene promoter. Reporter gene constructs driven by different promoter and enhancer regions of the Pax6 gene were generated and used to assay the relative strength of different Pax6 gene promoter regions. QRT-PCR and Wes were used to analyze mRNA and protein expression levels.
Results :
RNAseq analysis revealed the reverse relationship between CREB and Pax6. EMSA assays demonstrate that CREB directly binds to the promoter region of Pax6. ChIP assay results confirmed that CREB binds to Pax6 promoter in vivo. In mouse lens epithelial cell, overexpression WT-CREB resulted in downregulation of Pax6. Silence of CREB upregulates Pax6. RNAseq analysis also revealed that CREB can regulate a panel of differentiation-related genes.
Conclusions :
Through direct control of Pax6 and other downstream differentiation-related genes, CREB regulates lens differentiation. Supported by National Natural Science Foundation of China (Grants #82000842, #81970787, #82000876, #81770910) and Natural Science Foundation of Guangdong Province and Guangdong City Joint Program of China (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.