Deciphering the molecular network surrounding ZNF408 in the regulation of retinal angiogenesis

Abstract

Purpose : A missense mutation in ZNF408 (p.His455Tyr) was reported in a family with an autosomal dominant form of familial exudative vitreoretinopathy (FEVR). ZNF408 encodes a protein that belongs to the zinc finger transcription factor family. Previous in vitro and in vivo studies implied the pathogenicity of the mutation and revealed an important role for ZNF408 in the development of vasculature. Nonetheless, little is known about the downstream transcriptional targets of ZNF408 and how it contributes to (retinal) angiogenesis.

Methods : The molecular mechanisms of ZNF408-associated FEVR were investigated in vitro by overexpression of wild-type and mutant ZNF408 in cellular systems. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) was employed to reveal the genome-wide ZNF408 binding sites. Subsequently, RNA sequencing (RNA-Seq) in a vascular endothelial cell line was performed to determine the genes whose expression is dependent on ZNF408.

Results : The data obtained by ChIP-SEQ showed that ZNF408 binds predominantly to promoter regions of genes involved in various processes, including, but not limited to, protein localisation, macromolecular complex assembly, and RNA processing. Interestingly, the data obtained also showed that the mutant ZNF408 has a reduced ability to bind to its targets. These findings were validated by luciferase-based transactivation assays on a selected subset of targets. Transcriptome analysis revealed distinct gene expression profiles in cellular models overexpressing wild-type ZNF408 compared to mutant ZNF408. Furthermore, the transcriptome data allowed the identification of genes whose expression is dependent on ZNF408.

Conclusions : The combination of the ChIP-Seq and RNA-Seq datasets allowed the identification of the transcriptional targets of ZNF408. It also allowed us to clearly distinguish the differential role of wild-type versus mutant ZNF408 in the regulation of transcription in the context of (retinal) angiogenesis. Finally, the identification of the transcriptional targets of ZNF408 may reveal novel candidate genes for unsolved FEVR cases and thereby increase our understanding on the molecular mechanisms underlying FEVR.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.