INVESTIGATION OF DNA SECONDARY STRUCTURES AS DRIVERS OF ALTERED EPIGENETIC MODIFICATIONS IN OBESITY AND TYPE 2 DIABETES

Overweight and obesity represent a major health issue of the 21 century. According to the WHO, the worldwide rate of obesity has tripled from 1975 to 2016. Alarmingly, also childhood obesity increased from 0.7-0.9 % in 1975 to 5.6-7.8 % in 2016, with a higher prevalence in boys than girls. The excessive accumulation of fat increases the risk of developing comorbidities such as type 2 diabetes, the metabolic syndrome, and cardiovascular diseases, as well as gastric, pancreatic and colorectal cancers, among others. Type 2 diabetes mellitus (T2D) is characterized by a combination of reduced insulin sensitivity of cells and a decrease in pancreatic β-cell insulin secretion. While T2D is the most common type in adults, particularly the number of diagnoses in young people is increasing at an alarming rate. The imbalance between caloric input and output leading to obesity and with this a high risk for T2D is owed to our dietary habits, physical inactivity, stress, sleep patterns, social and educational status, endocrine disorders or medication such as corticosteroids. Genome wide association studies (GWAS) have also identified risk loci, however, such genetic risk loci cannot fully explain the observed variance in disease risk. Hence, other mechanisms beside the alterations in the genetic code have to be taken into account as potential drivers of the disease. Since obesity and T2D are strongly dependent on environmental and life style factors, alterations in epigenetic patterns present a powerful explanation for disease development and progression. Epigenetic modifications, including complex changes and interactions in DNA methylation, histone modifications and non-coding RNA expression, can directly influence gene expression without primary changes in the genetic code. In contrast to genetic changes, epigenetic changes are reversible, which offers interesting therapeutic perspectives.

Since the first epigenetic study with T2D in 2008, multiple genes have been shown to be affected by alterations in DNA methylation in patients with obesity and T2D versus controls. Currently, not much is known about the exact processes influencing DNA methylation, but a very recent idea is, that even the DNA structure, if it is a β-Helix or it forms diverse secondary (non-B DNA) structures, may influence DNA methylation. Therefore, the purpose of this project is to analyze for the very first time, if the observed changes in DNA methylation in obesity and type 2 diabetes are potentially linked to changes in DNA secondary structure formation. Specifically, we will 1) investigate the influence of high-energy supply (glucose and fat) on non-B DNA maps; 2) investigate if DNA methylation is influenced by the non-B DNA structures, especially at genes identified in epigenetic studies with obesity and T2D patients; and 3) by selectively influencing the formation of non-B DNA structures or DNA methylation with epidrugs, we sought to gain insight on the response of gene expression. This is of high relevance for further investigations, if epigdrugs and ligands targeting non-B DNA structures could be a potential treatment for T2D.

In order to address these aims, we propose to generate genome-wide non-B DNA maps with an Illumina-based next generation sequencing method called Permanganate/S1 footprinting (Perm-Seq) in insulin-sensitive HepG2 human liver cell line, as well as in the insulin-secreting EndoC-βH1 pancreatic islet cell line. The methylation status at regions of interest identified from epigenetic studies in diabetes and obesity will be determined with bisulfite conversion coupled to pyrosequencing. A wide range of techniques such as Western Blots, mRNA expression profiling, qPCR, ssDNA sequencing and bioinformatics analysis are required to unravel the mysteries surrounding the role of non-B DNA structures in epigenetic regulation of metabolic diseases.

This study will, for the very first time, shed light on mechanisms influencing certain epigenetic processes. This is of high relevance with regard to developing new treatment strategies. Epidrugs have evolved as important treatments for cancers such as acute myeloid leukemia (AML) and probably could also move forward to diabetes therapy.