Abstract
Purpose :
Glaucomatous trabecular meshwork (GTM) cells retain a pathological phenotype upon extraction and in vitro culture. Evidence suggests that this involves gene expression and chromatin remodeling. However, the regulatory mechanisms are incompletely understood. Here, we use paired RNA/ATAC-seq analysis to characterize the transcriptomic/epigenomic profile in GTM vs. normal TM (NTM) cells in a tissue-like soft ECM environment.
Methods :
NTM or GTM cells (N=5 strains each) were grown on soft tissue-like 3D ECM hydrogels. Cells were harvested for RNA/nuclei extraction to prepare RNA/ATAC-seq libraries. Genome-wide expression profiling was done using 100 bp paired-end RNA sequencing; data was processed using established Partek-Flow pipelines (alignment to hg38 human genome, four co-factor DESeq2 differential gene expression analysis [glaucoma status, TM cell prep laboratory, donor sex and age], p-value cut-off of 0.01, fold-change of ≥2.0). Chromatin accessibility mapping was done using 50 bp paired-end ATAC sequencing; data was again processed using Partek-Flow (calling sequencing peaks, filtering peaks with -log10(pvalue)<10, annotating peaks for TSS plot generation, DESeq2 analysis of differentially altered genomic peaks in GTM vs. NTM cells including the four co-factors).
Results :
RNA-seq analysis identified 170 differentially expressed genes in GTM cells vs. NTM cells. Gene ontology analysis showed enrichment of genes involved in collagen fibril organization, cell chemotaxis, endopeptidase inhibitor activity, ECM (collagen fibril) organization, and ECM-receptor interaction. ATAC-seq analysis identified 600 differentially altered open chromatin regions in GTM cells vs. NTM cells. Bioinformatics analysis showed enrichment of genes involved in cell migration, regulation of cell differentiation, caveola, focal adhesion, cell adhesion molecule binding and histone deacetylase complex, with 16 differentially expressed genes displaying significantly altered open chromatin regions: ABTB2, ARID5A, ATP2A2, CTD-3088G3.8, DPYSL2, FAP, MSC-AS1, NEAT1, RP11-557N21.1, SCX, SMURF2, SSR3, SYNGR3, VEPH1, WNT4, and ZNF295-AS1.
Conclusions :
Our data suggest distinct transcriptomic and epigenomic profiles in glaucomatous compared to normal TM cells, with candidate genes providing novel evidence of potential connections between altered open chromatin regions and nearby gene expression.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.