Chromatin access regulates the formation of Müller glia-derived progenitor cells in the retina

Abstract

EZH2 (Enhancer of Zeste 2), a key component of the Polycomb Repressor Complex 2 (PRC2), is required for the differentiation of Muller glia (MG) during retinal development. However, nothing is known about the roles of EZH2 and PRC2 in mature or damaged retinas. Herein, we investigate the expression of PRC2-related factors and roles during the formation of Muller glia-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, EZH2, PRC2-related factors, and EZH2-activators, JARID2 and AEBP1, are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of EZH2, EED or JARID2 blocks the formation of MGPCs. Similarly, inhibition of EZH2 suppresses the proliferation of progenitors in the circumferential marginal zone. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with EZH2 inhibition and NMDA-treatment, and many of these genes are associated with glial and neuronal differentiation. Correlation across gene expression, chromatin access and transcription factor motif access in MG was observed for transcription factors known to covey glial identity and promote retinal development. In the mouse retina, components of the PRC2 complex are downregulated in MG in damaged retinas and inhibition of EZH2 has no effect upon the formation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick, but not mouse, the activity of EZH2 is required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

ID factors regulate the ability of Müller glia to become proliferating neurogenic progenitor‐like cells

“…We further analyzed expression of ID factors in a scRNA‐seq database that we generated for early time‐points, 3 and 12 h, after NMDA‐treatment (Campbell et al, 2023; Campbell, Blum, et al, 2021) (Figure 2h). These scRNA‐seq libraries were generated with different reagents and sensitivities.…”

“…To assess changes in chromatin access we probed scATAC‐seq libraries of retinas treated with saline or NMDA at 24 h after treatment that were generated and described in our previous study (Campbell et al, 2023). UMAP embedding of cells revealed distinct clusters of different types of neurons and glia (Figure 10a,b).…”

“…For scATAC‐seq analysis, including motif enrichment and motif scoring, all reference or annotation genomes were in NCBI/Ensembl format. The original detailed analysis of these scATAC‐seq libraries are described elsewhere (Campbell et al, 2023).…”

Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas

“…We performed analyses on MG from each treatment group and compared changes across the most significant LRinteractions (Fig. 2A-D To identify putative paracrine LR-interactions between MG and microglia, we bioinformatically isolated MG and microglia from scRNA-seq libraries that we previously generated and described in detail (Campbell et al, 2021b;Campbell et al, 2022;Campbell et al, 2023). These libraries were generated from cells captured from normal and NMDA-damaged retinas at 3, 12 and 48hrs after treatment (Fig.…”

ID transcription factors regulate the ability of Müller glia to become proliferating neurogenic progenitor-like cells

“…For scATAC-seq analysis, including motif enrichment and motif scoring, all reference or annotation genomes were in NCBI/Ensembl format. The original detailed analysis of these scATAC-seq libraries are described elsewhere (Campbell et al, 2023).…”