Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Leaching of the plastic constituents leading to their chronic exposure to humans is a major concern for our environmental and occupational health. Our previous and other numerous studies have demonstrated that environmental chemicals like di (2-Ethylhexyl)-phthalate (DEHP) could pose a risk towards the epigenetic mechanisms. Yet, the mechanisms underlying its possible epigenotoxicity are poorly understood. We aimed to assess the impact of DEHP exposure to the human breast cancer cells (MCF-7) and resultant changes in DNA methylation regulators ultimately altering the expression of the cell cycle regulator p21 as a model gene. The MCF-7 cells were exposed to environmentally relevant concentrations (50-500 nM) for 24 h. The results showed that DEHP was proliferative towards the MCF-7 cells while it induced global DNA hypermethylation with selective upregulation of DNMT1 and MECP2. In addition, DEHP significantly reduced p53 protein and its enrichment to the DNMT1 promoter binding site, while elevating SP1 and E2F1 transcription factor levels, stimulating their binding to the promoter DNA. Coincidently, increased DNMT1 level was highly associated with loss of p21 expression and increased cyclin D1 levels. Importantly, the p21, but not cyclin D1 promoter CpG-dinucleotides were hypermethylated after exposure to 500 nM DEHP for 24 h. Furthermore, it was observed that DEHP significantly enriched DNMT1 and MECP2 to the p21 promoter to induce DNA methylation-based epigenetic silencing of p21, resulting in increased cell proliferation. Our results suggest DEHP could potentially induce the epigenetic alterations that might increase the risk of breast cancer, given that the underlying mechanisms should be fully elucidated.

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root-evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation-qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.