EZH2 and the polycomb repressor complex 2 are required for the reprogramming of Müller Glia into progenitor cells in the retina.
MetadataShow full item record
Series/Report no.:2021 Edward F. Hayes Graduate Research Forum. 35th
Purpose: Many retinal diseases lead to the loss of retinal neurons, resulting in significant visual impairment and blindness. In mammals, there is no regenerative response to replenish lost neurons and restore vision. However, retinal regeneration occurs in chick and zebrafish where lost neurons are regenerated. Müller glia, the primary glial cell of the retina, are the source of these regenerated neurons. Müller glia detect retinal damage, de-differentiate into progenitor cells, and progeny differentiate to become neurons. The transition from glia to neuron requires reorganization of chromatin architecture, which is accomplished through epigenetic regulation and histone modifications. The polycomb repressor complex 2 (PRC2) is a histone methylase that regulates retinal development, and knockout of the catalytic subunit EZH2 results in microphthalmia. Thus, we hypothesize that the PRC2 complex is necessary for the repression of glial fate genes, permitting Müller glia to de-differentiation and become progenitor-like cells. Therefore, we investigated the epigenetic regulation of Müller glia reprogramming. Research Methods: Postnatal White Leghorn chicks (P7-14) were intravitreally injected with excitotoxin N-methyl-D-aspartate (NMDA) to induce retinal damage and initiate Müller glia reprogramming. We administered EZH2 inhibitor 3-Deazaneplanocin A (DZN) to determine the impact on the formation of proliferating Müller glia derived progenitor cells. We enucleated, fixed, and sectioned the tissue for fluorescence immunohistochemistry (IHC). We quantified the number of progenitors that entered the cell cycle by staining for the incorporation of nuclear 5-Ethynyl-2'-deoxyuridine (EdU) to colocalize with Müller glia/progenitor markers Sox2/Sox9. Retinal preparations were dissociated for single cell analysis. We performed single cell RNA sequencing (scRNA-seq) and a single cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) on damaged, undamaged, and DZN treated retinas to determine how changes in chromatin remodeling and gene expression occurred during reprogramming and were influenced by inhibition of EZH2. Results: We found that EZH2 and other components of the PRC2 complex are specifically upregulated in Müller glia during the de-differentiation process to form Müller glia derived progenitor cells. Single cell sequencing indicates an upregulation of EZH2, EED, SET and RBBP4. IHC of EZH2 protein also colocalized with Müller glia nuclei after damage. The inhibition of EZH2 with DZN blocked the formation of progenitor cells after damage. scRNA-seq analysis demonstrated that EZH2-inhibition significantly influenced gene expression and chromatin access. This led to increased expression in glial transcription factors (ID4, NFIA, SOX8) in damaged retinas, while also increasing reactive glial genes (ATF4, NFIX, NEFM, DCX, HES1). When observing chromatin accessibility in scATAC-seq data, DZN treatment increased access to many important genes, including pro-glial factors such as ID4 and NFIX. Genomic regions surrounding both glial (NOTCH1, GLI2) and neuronal (HEYL1, LHX9) transcription factors were also more accessible as a result of inhibiting the PRC2 complex. Implications: We find that PRC2 complex genes are upregulated in Müller glia-derived progenitors during reprogramming. We demonstrate the pharmacological inhibition of EZH2 robustly negates the transition from glia to progenitor cell after damage. Single cell analyses of chromatin accessibility indicate that this repressive histone methylase condenses chromatin around transcription factors that promote mature glial or neuronal phenotype. These epigenetic modifications correlate to the increased expression glial genes the repress a progenitor cell phenotype. We identify genes that may be key regulators of Müller glia's response to damage and are required for the transition into progenitor cells. These findings provide new insight into how epigenetic factors impact regeneration in the retina and central nervous system. Future experimentation is required to study how histone modifications influence the poor regenerative response in the retinas of mammals.
Biological Sciences: 3rd Place (The Ohio State University Edward F. Hayes Graduate Research Forum)
Items in Knowledge Bank are protected by copyright, with all rights reserved, unless otherwise indicated.