Fig. 2 | Scientific Reports

Fig. 2

From: G-quadruplex forming regions in GCK and TM6SF2 are targets for differential DNA methylation in metabolic disease and hepatocellular carcinoma patients

Fig. 2

Non-B DNA formation in GCK and TM6SF2: (A). Shows GCK (chr7: 44,184,901–44,187,919, hg19), TM6SF2 (chr19: 19,380,358–19,382,362, hg19) and the non-G4 forming region in PARD3B (chr2:205,780,593–205,782,891, hg19). Data from different sources are shown in individual rows: Pink rectangles indicate R-loop forming sequences (RLFS) calculated by Quantitative Model of R-loop Forming Sequence (RLFS) finder (QmRLFS-finder)76. Blue rectangles represent putative G4 forming motifs (PQS) obtained from pqsfinder77, Black track: in vitro G4 formation stabilized with pyridostatin (PDS) in HEK293T cells78; Grey track: ChIP input control79; Red tracks: G4-ChIP-Seq tracks in HEK293 cells (2 replicates)79; Blue tracks: PDAL-Seq replicates 1 and 2 of HEK293 and MCF7 cells, Olive tracks: transcription factor EGR1 binding in K546 cells82. The sequences within GCK (antisense G4, sequence is given as reverse complement) and TM6SF2 (sense G4) forming a G4 structure are detailed below. G-tracks are underlined, grey boxes indicate CpG positions. Blue highlighted areas: identified differentially methylated regions; light red highlighted area: a second regulatory region in GCK, which can form G4s. (B). Native DNA PAA gels identify in vitro G4 formation in the G4 control region in MYC, GCK exon 7 and in the TM6SF2 intron 2-exon 3 boundary, but not for ESR1 (negative control). PCR products are loaded as unfolded (50 mM Tris–HCl pH 8.3) (U), folded with 150 mM KCl and heat induction (K), and folded with 150 mM KCl, heat induction and 10 µM PDS (PDS). (C). Positive and negative peak maxima obtained from CD spectra for MYC, GCK and TM6SF2 with increasing K + concentrations, for the methylated or mutated oligos. Individual CD spectra are shown for (D). MYC, (E). TM6SF2 and (F). GCK. (G). Depicts the possible refolding of the methylated hairpin to a parallel G4 structure caused by demethylation at the GCK exon 7 region. The green loop indicates the bulge sequence which interrupts G-run two.

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