Epigenetic and Ubiquitin-Proteasome Mechanisms of Obesity Development

Obesity is a major health condition in which little is known about the molecular mechanisms that drive it. The hypothalamus is the primary control center for controlling both food intake and energy expenditure in order to maintain the body's energy balance and dysregulation of molecular processes in this region have been implicated in the development and progression of obesity. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which tracked body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed. Importantly, CRISPR-dCas9 mediated upregulation of DNA 5-hmC enzymes in the male, but not female, hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. Next, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Moreover, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Finally, we found that both males and females showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. However, while the CRISPR-dCas9 system was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. Collectively, this data reveals novel, sex-specific differences in the engagement of the ubiquitin proteasome system and role of DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype.