Functional Dissection Shows FOXE3 Contributes to Peters Anomaly through the Transcriptional Regulation of an Autophagy Associated Co-Chaperone, DNAJB1

Abstract

Purpose : We previously identified a familial case of Peters anomaly (PA) harboring a nonsense mutation (p.C240*) in FOXE3. The current study was undertaken to identify the downstream targets of FOXE3, which are differentially expressed as a result of the premature termination of FOXE3.

Methods : The wild-type and mutant FOXE3 (p.C240*) were expressed in HEK293FT cells, and their respective transcriptome and proteome were determined through next-generation RNA sequencing (RNASeq) and mass spectroscopy based protein sequencing (ProSeq), respectively. The datasets of RNASeq and ProSeq analysis were investigated for differentially expressed candidates. The expression of potential candidate genes was knocked down using shRNA- and morpholino-based methodologies in human lens epithelial cells (HLE) and zebrafish embryos, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was employed to investigate apoptosis in HLE cells and morphant embryos. Likewise, propidium iodide staining was used to examine cell cycle defects in HLE cells while the histological analysis of morphants embryos was completed by hematoxylin and eosin (H&E) staining.

Results : We identified DNAJB1, an autophagy-associated co-chaperone that is highly expressed in ocular lens, as the sole candidate exhibiting differential expression in both transcriptome and proteome screens. We confirmed the candidacy of DNAJB1 through ChIP and luciferase assays while knockdown of DNAJB1 resulted in the mitotic arrest and increased apoptosis in HLE cells. Subsequently, splice-site, and translation blocking morpholinos were used to knockdown dnajb1 (dnajb1a and dnajb1b) in zebrafish embryos, which resulted in underdeveloped cataractous lenses with persistent apoptotic nuclei. The histological examination of zebrafish morphants illustrated smaller eyes with an elongated lens, deteriorated cornea and distorted angle mesenchyme recapitulating symptoms of PA. TUNEL staining of 4dpf zebrafish embryos show increased apoptotic nuclei in the morphant lens compared to normal embryos.

Conclusions : In conclusion, we have identified DNAJB1 as a downstream target of FOXE3 through a combination of transcriptional and proteomic approaches that was subsequently validated through in vitro and in vivo assays. These data emphasize the role of autophagy in the development of the ocular tissue.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.