Abstract
Purpose :
Heterochromatic epigenetic modifications have previously been shown to restrict the potential of neural stem cells, but it has remained unclear how these processes are controlled. Using retina as a model system, our lab identified that transcription factor Casz1 interacts with Chd4 nucleosome remodeller to regulate the potential of retinal progenitor cells (RPCs) during development. We hypothesize that the Chromodomain-helicase-DNA-binding-4 (Chd4) regulates RPC potential by restricting the epigenome. Our objective is to determine whether and how Chd4 regulates RPC potential.
Methods :
Chd4 contains an ATPase-Helicase domain which utilizes energy from ATP hydrolysis to perform the nucleosome remodelling activity. We obtained a floxed Chd4 allele, that was cross-bred with a retina-specific Chx10- Cre allele to excise the enzymatic region of the Chd4 gene specifically in the developing retina. We first performed phenotypic analysis to identify the role of Chd4 during retinal development and follwed up with "omic" studies to identify the mechanisms that might regulate RPC potential.
Results :
Phenotypic analysis of adult retinas showed that conditional knock-out (cKO) of Chd4 lead to an atypical retinal architecture with highly distorted retinal layers as compared to control. Using cell-specific markers we ascertained that Chd4 cKO lead to a skewed generation of different retinal cell-fates, where early cell types such as retinal ganglion cells and horizontal cells were overproduced at the expense of late-born cell-fates such as rod photoreceptors. Additinally, through Edu birthdating it was observed that Chd4 is required for proper temporal transitions in RPC potential. To determine how Chd4 affects gene expression, we performed single-cell RNA-seq on control and cKO RPCs. Through our preliminary analysis of this dataset, we have been able to identify many genes that are differentially expressed in cKO RPCs and are currently in the process of validating these data. Furthermore, we performed ATAC-seq on control and cKO RPCs to determine how Chd4 regulates gene accessibility to modulate RPC potential, and, observed that Chd4 cKO lead to an increase in genomic accessibility.
Conclusions :
We propose a working model where Chd4 is required in the developing retina to restrict genome accessibility as development continues, so as to limit the generation of early cell fates and promote proper temporal transitions in RPC potential.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.