ASSESSING DNA METHYLATION PATTERNS AT PROMOTOR REGIONS OF GROWTH PLATE GENES FOLLOWING GESTATIONAL ENVIRONMENTAL AND NUTRITIONAL STRESSORS

Abstract

LGestational exposure to adverse environmental and nutritional stressors profoundly influences fetal programming and long-term skeletal development. While epidemiological and animal studies link prenatal adversity to impaired longitudinal bone growth, the precise cell-autonomous epigenetic mechanisms occurring at the growth plate remain poorly defined. To investigate these mechanisms independently of whole-organism systemic confounders, we utilized an in vitro human epiphyseal chondrocyte progenitor model exposed to combined nutritional (one-carbon methyl-donor restriction: folate/methionine deficiency) and environmental (mild hypoxia, 3% O2 stressors mimicking adverse intrauterine milestones. Targeted bisulfite sequencing and quantitative real-time PCR were employed to assess DNA methylation patterns at CpG islands within promoter regions of master chondrogenic and longitudinal growth-regulating genes (SOX9, COL2A1, ACAN, and IGF1R). Our findings reveal significant, locus-specific hypermethylation at the promoter regions of SOX9 and IGF1R following stress exposure, accompanied by a marked downregulation of transcript expression. Conversely, housekeeping controls and non-target loci remained stable. These in vitro results demonstrate that gestational stressors directly induce site-specific epigenetic modifications in chondrogenic progenitor cells, providing a mechanistic bridge between early-life adversity and compromised skeletal growth phenotypes.