Epigenetic deregulation of the HNF1A gene in diabetes and pancreatic adenocarcinoma
(funded partially through HRZZ project EpiGlycoIgG)
Molecular mechanisms leading to diabetes are not yet fully understood. It is know from studies on mice that high fat diet decreases expression of the HNF1A gene, which is normally highly expressed in liver and pancreas. The transcription factor HNF1A is involved in glucose uptake and proper GSIS (Glucose Stimulated Insulin Secretion) through regulation of the glyco-gene MGAT4A. This gene encodes for glycosyltransferase GntIV which is responsible for correct glycosylation of the Glut transporter on β cells and its localization at cell surface, where it mediates glucose uptake. Similar mechanism is supposed to occur in human Langerhans islets. Our previous results have revealed the correlation between DNA methylation from blood and plasma glycans in patients with HNF1A-MODY patients (a subtype of type 2 diabetes caused by HNF1A mutation) (Zoldoš et al. 2012 Epigenetics).
It has been postulated that a common comorbidity of diabetes is pancreatic adenocarcinoma. The aim of our research is to analyze CpG methylation in the HNF1A gene promoter in β cells of Langerhans islets in pancreas of people who had adenocarcinoma and were previously diagnosed with diabetes. Methylation analysis will be performed using pyrosequencing after bisulfite conversion of DNA extracted from microdissected pancreatic tissue embedded in paraffin. Total glycans from β cells of Langerhans islets of normala tissue as well as in tumor tissue will be determined using Nano LC-MS in order to establish whether HNF1A gene regulates N-glycosylation in diabetes and pancreatic cancer, respectivelly. In addition, putative regulatory sites in the HNF1A promoter will be modulated using CRISPR/Cas9-DNMT3A tool in HCC tumor cell line, the HepG2. N-glycans from secreted glycoproteins will be analyzed using HILIC and MS by our partners.
Comprehensive Toolbox for Epigenetic Modulation of Gene Expression
The CRISPR-Cas9 technology enables targeting of almost any sequence in the genome and has been extensively used for genome editing (i.e. gene knock-outs). We have created a Cas9-based tool for epigenetic silencing of eukaryotic gene expression by targeted DNA methylation, where the Cas9 nuclease has been repurposed by fusion to a DNA methyltransferase 3A (DNMT3A) catalytic domain (Vojta et al. 2016 Nucleic Acids Research). As a follow-up to our efforts, we are creating a comprehensive molecular toolbox harnessing excellent targeting properties of Cas9 and using it to deliver domains for activation, repression, DNA methylation/demethylation and histone modifications to promoters or other control elements of targeted genes. The molecular toolbox will be highly modular and easily reconfigured for a range of applications in gene regulation and epigenome editing. We aim to apply the toolbox to resolve biological questions from our ongoing research ranging from studies of metabolic pathways of protein N-glycosylation to studies of mechanisms behind human complex diseases such as HNF1A-MODY subtype of diabetes, inflammatory bowel disease and amyotrophical lateral sclerosis.
Figure: The CRISPR/Cas9-DNMT3A tool for targeted CpG methylation: single guide RNA (sgRNA) is in complex with the recognition lobe (RecI, II and III domains) and the nuclease lobe (HNH, RuvC and PI domains) of the Cas9 protein. The C-terminus of the Cas9 protein is located within the PAM interacting (PI) domain and faces the side where the bound genomic DNA protrudes with its 3’end relative to sgRNA sequence. The catalytic domain of DNMT3A recruits it’s partner for dimerization DNMT3L in vivo.
Integrated approach to social and economic reintegration of drug addicts
(The project is funded through IPA-CLOUD project; collaboration with prof. Darko Roviš, and prof. dr. sc. Tihana Lenac Roviš, Center for proteomics, Faculty of Medicine University of Rijeka, Croatia)
Genetic and epigenetic variability in the serotonin transporter gene, the SLC6A4, has been previously linked with vulnerability to stress-related psychopathology upon exposure to environmental adversity. This project deals with genotyping the adolescents with risk behaviour (drug addiction) for the SLC6A4 gene represented in the human population with three alleles – S, LG and LA – of which alleles S and LG are associated with predisposition to drug addiction. Our group aims to study DNA methylation profile within the SLC6A4 gene in the cohort of young addicted individuals compared with healthy individuals.