ROLE OF DNA METHYLAION IN THE PROMOTER REGION OF CLOCK GENES IN THE REGULATION OF LIVER DISEASE

Background and aim Obesity is becoming a major global health concern, as it is associated with many diseases, such as Non‐Alcoholic Fatty Liver Disease (NAFLD). NAFLD is associated with diabetes, metabolic syndrome and, and is known to be multifactorial. Meaning that it is affected by multiple external and environmental factors. The progression of NAFLD to Non‐alcoholic Steatohepatitis (NASH), liver carcinoma and further to liver failure, makes it very crucial that we pay attention to the disease at its earlier stages. Further, NAFLD is affected by the circadian rhythm, which is a 24‐hour cycle that controls biological activities of all cells. In the liver, this rhythmic cycle is called liver clock and is very sensitive to nutritional intake, physical activity and sleeping patterns, and it is controlled by Clock genes. The expression of clock genes can be controlled by DNA methylation, which is a modification that acts as a switch to turn genes on and off. Therefore, this study aimed to study the methylation status of clock genes and clock‐controlled genes at the promoter region. Methods C57BL/6 mice were treated with high‐fat diet (HFD) or control (CTR) for 8 wk. Mouse livers were harvested for further analysis. Liver function was evaluated by serum chemistry, fibrosis was measured by IHC and PCR and lipid deposition were evaluated by Oil red O staining. Genomic DNA and total RNA were obtained for measuring the DNA methylation of core clock genes and RNA expression as described above. Methylation‐Specific PCR (MSP) and Sanger sequencing were used to evaluate the change in the gene expression of genes of interest; CLOCK, BMAL‐1 and PPAR‐a in a high‐fat diet group and control‐diet group. Results We found increased steatosis and hepatic fibrosis in mice fed a high‐fat diet compared to control. Expression of fibrotic markers (a‐SMA, FN1 and collagen I) was upregulated in mice fed high‐fat diet compared to control. Differential DNA methylation status in promoter regions was observed in different core clock genes in high‐fat‐fed mice. Conclusion DNA methylation of core clock genes and clock‐controlled genes may lead to dysregulation of the circadian rhythm during consumption of a high fat diet, thus leading to increased steatosis and liver fibrosis.