Abstract
Purpose :
Heterochromatin alterations is a hallmark of aging process. This study is aimed to investigate structure and distribution of heterochromatin on retinal pigment epithelial (RPE) cell premature senescence induced by oxidative stress exposure or X-ray irradiation, and to explore the effects of heterochromatin in regulating senescence-associated secretory phenotype (SASP) gene expression during RPE senescence.
Methods :
Heterochromatin structure was assessed by confocal microscopy and transmission electron microscope. Genome-wide heterochromatin distribution was studied by chromatin immunoprecipitation (ChIP)-seq analysis. ARPE-19 cells were continually exposed to tert-Butyl hydroperoxide (t-BHP) or X-ray irradiation to induce cell senescence, which was confirmed by senescence-associated ß-galactosidase (SA ß-gal) staining, senescent-associated gene expression and RPE barrier function analysis. Chromatin structure on SASP gene locus was assessed by formaldehyde-assisted isolation of regulatory elements (FAIRE), and the occupancy of heterochromatin there was determined by quantitative ChIP (q-ChIP) analysis. Heterochromatin was disrupted by chaetocin treatment. Expression of inflammatory cytokines were examined by qRT-PCR and cytokine protein array.
Results :
Heterochromatin mark H3K9me3 was enriched in SASP gene locus. A prominent nuclear peripheral heterochromatin loss was found in senescent RPE cells. Decreased chromatin compaction was detected in proinflammatory genes upregulated during RPE senescence. Disruption of heterochromatin led to robust upregulation of SASP genes.
Conclusions :
Heterochromatin represses SASP gene expression during RPE senescence. Our data suggest a potential role for targeting heterochromatin in the treatment of retinal diseases related to RPE senescence, such as age-related macular degeneration (AMD).
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.