Abstract
Purpose :
We have previously shown that Leukemia inhibitory factor (LIF) is induced in Müller cells as an endogenous protective neurokine capable of protecting retinal photoreceptors from both acute and chronic injury. Therapeutic administration of LIF protein or gene therapies can prevent the progression of inherited retinal degeneration in vivo. LIF-induced protection is exclusively mediated by the JAK/STAT3 pathway. STAT3 is a DNA-binding transcription factor also shown to regulate cell survival outside of the retina. Our study aims to elucidate the regulatory molecular networks associated with LIF signaling in ocular tissues and to understand the role of STAT3 activation in these processes.
Methods :
Groups of wildtype and Chx10-cre STAT3 knock-out mice, both equal male and female in each group, were intravitreally injected in 1 eye with 2 micrograms of LIF protein, and the contralateral eyes were injected with PBS as a control. The retinas were removed 2 days post-treatment and single nuclei were prepared with 0.01% digitonin. The single nuclei suspension was processed for single nuclei assay for transposase-accessible chromatin sequencing (scATACseq) using a 10x Genomics microfluidics system. DNA libraries were sequenced, and chromatin accessibility profiles were identified in all retinal cell types using the R software, Seurat, and Signac packages.
Results :
We analyzed 42713 nuclei with an average of 10726 reads per cell. We identified 736 chromosomal regions associated with 630 distinct genetic loci exhibiting changes in chromatin accessibility pattern in LIF-treated retinas with an average log2 fold change of at least 0.5. For quality control, we identified the open chromatin genes known to be associated with LIF signaling: Fgf2 (4.82-fold difference), Edn2 (1.52), Stat3 (1.68), Socs3 (1.46), Bcl3 (1.62), Lifr (1.52). Chromatin accessibility was compared to scRNAseq data to determine chromatin accessibility changes associated with STAT3 activation to identify potential STAT3-regulated genes. Open chromatin regions were analyzed by motif analysis to identify transcriptional networks for regulating protective gene responses.
Conclusions :
Single nuclei ATACseq of LIF-treated retinas can be used to identify potential transcriptional network mechanisms of LIF-mediated protection.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.