RESUMO
Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.
RESUMO
Long non-coding RNAs transcribed from telomeres, known as TERRA (telomeric repeat-containing RNA), are associated with telomere and genome stability. TERRA abundance responds to different cell stresses; however, no studies have focused on oxidative stress, condition that damages biomolecules and is involved in aging and disease. Since telomeres are prone to oxidative damage leading to their dysfunction, our objective was to characterize TERRAs and the mechanisms that control their expression. TERRA increased in cells exposed to H2O2 and reverted by antioxidant treatment. TERRAs are also induced in brown adipose tissue of mice exposed to cold, which raises mitochondrial ROS. In cells exposed to H2O2, ChIP showed that chromatin landscape was modified favoring telomere transcription. TERRAs interacted with HP1α/γ, proteins that were found recruited to subtelomeres. Since HP1γ interacts with the transcriptional machinery, TERRAs may stimulate their own expression by recruiting HP1γ to subtelomeres. TERRA induction reverted within 2 h after removal of H2O2 from culture medium, suggesting they have protective functions. This was supported by rapid TERRA induction following a second H2O2 challenge. PKA inhibitors H89 and PKI blocked TERRA increase by H2O2 or IBMX+Forskolin treatment, suggesting PKA signaling regulates TERRA induction. Treatment of cells with drugs that disturb cytoskeleton integrity or growing cells on surfaces of different stiffness known to generate differential cytoskeleton tension also modified TERRA levels and sensitized cells to lower H2O2 concentrations. In summary, we show that TERRAs are induced in response to oxidative stress and are regulated by PKA as well as by changes in cytoskeleton dynamics.