Periodic Reporting for period 1 - EASY (Epigenetic approach for the treatment of obesity) Período documentado: 2017-07-01 hasta 2019-06-30 Resumen del contexto y de los objetivos generales del proyecto Obesity is one of the greatest public health challenges of the 21st century. In 2016, 39% of the world’s adults were overweight, of which over 650 million adults were obese.Current treatments mainly aim at limiting energy intake, but have very little efficacy, implying the involvement of additional factors. Beside the genetic contribution to the disease, in the last years much attention has been given to epigenetic deregulation as major contributor to metabolic disorders. Epigenetic refers to heritable changes in gene expression (active versus inactive genes), which occur without alteration in DNA sequence. There is a strong interplay between environmental/genetic factors and epigenetic changes in the establishment of obesity. Genes and environment can interact through their influence on the epigenome, and epigenetic marks can directly lead to increased adiposity. Although epigenetic changes may cause obesity, it is often not really clear if they precede obesity, or vice versa. Adipose tissues (AT), classified as white (WAT) and brown (BAT) are key organs for metabolic disorders. WAT is the major energy store site of the body. Conversely, BAT is rich in mitochondria and has energy expenditure properties. Owing to its ability to dissipate energy as heat, BAT has attracted scientific interest as an antiobesity tissue.Preliminary data generated in the lab pointed toward a role for the epigenetic regulator Suv4-20h in improving BAT activity through epigenetic modification. Hence, my project aims at defining how Suv4-20h proteins influence metabolism regulation in response to environmental stimuli. Moreover, I want to identify and characterize the molecular mechanism(s) through which Suv4-20h proteins regulate adipogenesis. My hypothesis is that interfering with Suv4-20h activity in BAT results in increased metabolic activity and weight loss. Through in vivo, ex-vivo, in vitro and genome-wide studies we characterized the role of Suv420h proteins in the regulation of metabolism and body weight. We found that Suv420h proteins respond to environmental stimuli by directly inhibiting the expression of PPAR-γ, a master transcriptional regulator of glycemic metabolism, adipogenesis, energy balance and lipid biosynthesis. Mice lacking Suv420h proteins specifically in BAT display a strong PPAR-γ activation signature, increased BAT mitochondria respiration, improved glucose tolerance, reduction in AT and resistance to obesity. In addition, we found significant Suv4-20h1 and Suv4-20h2 upregulation and PPAR-γ downregulation in human obesity.To the best of our knowledge, Suv420h1/2 are the first HMTases inhibiting BAT metabolism. Moreover, we describe the first epidrug able to activate BAT metabolic activity. Our results promote Suv4-20h proteins as key epigenetic regulator of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli. Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha We generated double-KO mice lacking Suv420h1 and Suv420h2 in the interscapular brown adipose tissue (iBAT). Dko mice display reduced BAT mass compare to controls and increased mitochondria respiration, in line with an enhanced metabolic activity of dKO brown adipocytes. We found that dKO mice have significantly lower basal glycemia compared to controls, and tolerate the glucose overload significantly better than control mice in a glucose tolerance test (GTT). Also, dKO mice exposed to cold have no difficulty in maintaining their body temperature. To gain insight into the pathways affected by Suv420h deletion, we compared dKO to control iBAT using RNA-Seq. In line with the repressor activity of Suv420h proteins, Suv420h ablation resulted in a significant majority of upregulated genes. Functional Enrichment analysis for the upregulated genes returned significant enrichment for oxidative metabolism and mitochondrial function pathways we found also significant enrichment of genes related to proteasomal activity. We then evaluated how dko mice respond to diet-induced obesity (DIO). Over 14 weeks of high fat diet (HFD), control mice gained almost 50% more weight when eating HFD compare to dKO, and MRI quantification revealed smaller subcutaneous and perigonadal white adipose tissue depots (psWAT and pgWAT respectively) in dKO eating HFD compared to control mice. Histological analyses showed significantly reduced adipocytes size in both iBAT and pgWAT of dKO compared to controls. To identify gene expression changes that might account for dKO protection from DIO, we compared the transcriptomes of Suv420h-deficient to control iBAT. What caught our attention was the finding that upregulated genes in dKO BAT showed a significant enrichment in PPAR-γ signalling pathway. PPAR-γ is highly expressed in BAT and act as a master transcriptional regulator of glucose metabolism, adipogenesis, energy balance and lipid biosynthesis. Chromatin-immunoprecipitation experiments (ChIP) showed that Suv4-20h associated histone mark H4K20me3, is enriched at the PPAR-γ gene in proliferating pre-brown adipocytes, in which PPAR-γ is normally repressed. Treatment with the Suv420h specific inhibitor A-196 significantly abrogate H4K20me3 at the PPAR-γ gene and leads to a significant increase in PPAR-γ expression level compared to vehicle treated cells. These results suggest that PPAR-γ is a direct Suv420h target, which could play an important role in Suv420h metabolic regulation. Treatment of proliferating pre-brown adipocytes with A-196 is sufficient to significantly activate the expression of PPAR-γ direct target genes, which is blocked by concomitant treatment with a specific PPAR-γ inhibitor. Moreover, A-196 treatment leads to a significant increase of the respiratory rate of brown pre-adipocytes, and this effect depends on PPAR-γ since it is blocked by treatment with the PPAR-γ inhibitor. We have demonstrated that Suv420h ablation activates BAT metabolism, resulting in improved metabolic parameters and systemic protection against obesity. Our study provides the first demonstration of a direct role for Suv420h enzymes in the regulation of metabolism.Results have been presented to international conferences and during public events, and raised much interest among scientific community and general public given high therapeutic potential of our findings Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) It is emerging that both genetic and environmental effects on epigenetics are associated with metabolic diseases such as obesity. A better understanding of the gene(s) through which the effect is executed is necessary before any of these findings would lead to useful therapeutic interventions. Trough the development of the EASY project we provided a comprehensive understanding of the epigenetic mechanisms underlying energy balance regulation. We identified SUV420H proteins as key epigenetic regulators of glycemia and body weight in response to environmental stimuli, identifying the first epidrug able to activate BAT metabolic activity, which is disrupted in obesity. Results of our project are exploitable as potential therapeutic tools for treating human metabolic diseases, which will provide enormous benefit to world-wide population. White and brown adipose tissue in control and dKO mice upon HFD treatment
