G-quadruplex formation in GCK and TM6SF2 are targets for differential DNA methylation in metabolic dysfunction-associated fatty liver disease and type II diabetes mellitus patients

Background. The alarming increase in global rates of metabolic diseases (MetDs) and their association with an increased cancer risk renders them a considerable burden on our society. The interplay of environmental and genetic factors in causing MetDs may be reflected in DNA methylation patterns, particularly in non-canonical (non-B) DNA structures, such as G-quadruplexes or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intergenic regions of two MetD risk genes, glucokinase (GCK) exon 7 and transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which are overlapped by long non-coding RNAs. Results. Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. DNA methylation and gene expression data from The Cancer Genome Atlas (TCGA) for liver tumor versus normal tissue confirmed these observations. Detailed analysis including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. The newly established method permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), as well as standard methods such as native DNA gel electrophoresis and circular dichroism (CD) spectrophotometry confirmed the formation of G4 structures in these regions. Based on our analysis, we propose that the elevated blood glucose and fatty acid levels as observed in MetD patients could lead to reformation or topological changes in the non-B DNA landscape causing differential methylation and altered transcription factor binding. Conclusion. Our analyses provide a completely new view on the mechanisms underlying MetDs and their link to cancer, unveiling non-B DNA structures as novel targets for therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.