Ell2 regulates expression of key lens genes in development, perturbation of which causes early-onset lens defects

Abstract

Purpose : While several proteins that have a regulatory function in the lens have been characterized, key questions remain as to how lens cells produce abundant levels of specific mRNAs (e.g. crystallins). In this study, we addressed a provocative question by focusing on a regulator (Ell2 (Elongation factor for RNA polymerase II 2)) that is considered to be widely important for a general transcriptional process called “elongation” (involved in promoting transcription by relieving the transient “pause” of RNA Pol II), and examined whether its perturbation leads to specific transcriptional and phenotypic changes in the lens.

Methods : Ell2^cKO mice were generated using Pax6GFPCre and lens defects were characterized by microscopy and histology. Lens transcriptome was examined by RNA-sequencing (RNA-seq) and differentially expressed genes (DEGs) were validated by RT-qPCR. Chromatin immunoprecipitation (ChIP) was performed to determine occupancy of Ell2 on genomic regions in lens cells.

Results : Ell2 was identified as a highly lens-enriched gene by iSyTE and was also found to be misexpressed in mouse lenses deficient for other key regulators such as Celf1 and Elavl1 that are linked to cataract. Robust expression of Ell2 in mouse lens development was validated by RT-PCR, in situ hybridization, Western blotting and immunofluorescence and found to be high in fiber cells (FCs). In later postnatal stages, Ell2 was also detected in lens epithelial cells. Ell2 expression was also highly enriched in zebrafish lens development. Ell2^cKO mice exhibit fully penetrant lens defects starting at age 2 weeks as observed under light microscopy and histology. RNA-seq revealed reduced mRNA levels of a subset of FC genes including several crystallins, Actn2, Birc7, Dnase2b, Mip, etc. and AEL genes such as Adamts10, Aqp1, Dmrta2, Mapk14, etc. ChIP assay followed by genomic PCR on wild-type lens tissue shows that Ell2 protein is enriched at +100 bp region downstream of the transcription start site of key lens and cataract-linked genes that are also differentially expressed in Ell2^cKO lenses.

Conclusions : These findings suggest a novel paradigm that a “general” regulator of transcription, Ell2, can mediate “specific” expression control over select genes in lens (organ) development, in turn uncovering new mechanisms of regulating cataract-linked genes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.