Division of Cancer (Epi-)Genetics, Department of Biosciences and Medical Biology, Center for Tumor Biology and Immunology (CTBI), Paris Lodron University Salzburg, Hellbrunnerstraße 34, 5020, Salzburg, Austria.
Cancer Cluster Salzburg, Salzburg, Austria.
Sci Rep. 2024 Aug 30;14(1):20215. doi: 10.1038/s41598-024-70749-0.
The alarming increase in global rates of metabolic diseases (MetDs) and their association with 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 at non-canonical (non-B) DNA structures, such as G-quadruplexes (G4s) or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intragenic regions of two MetD risk genes, the glucokinase (GCK) exon 7 and the transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which harbor non-B DNA motifs for G4s and R-loops.Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. Furthermore, these regions harbor hypervariable and differentially methylated CpGs also in hepatocellular carcinoma versus normal tissue samples from The Cancer Genome Atlas (TCGA). Permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), native polyacrylamide DNA gel electrophoresis and circular dichroism (CD) spectroscopy revealed the formation of G4 structures in these regions and demonstrated that their topology and stability is affected by DNA methylation. Detailed analyses including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. Based on our analyses, we hypothesize that changes in DNA methylation lead to topological changes, especially in GCK exon 7, and cause the activation of alternative regulatory elements or potentially play a role in alternative splicing.Our analyses provide a new view on the mechanisms underlying the progression of MetDs and their link to hepatocellular carcinomas, unveiling non-B DNA structures as important key players already in early disease stages.
全球代谢疾病（MetD）发病率的惊人增长及其与癌症风险的关联，使它们成为我们社会的巨大负担。环境和遗传因素在导致 MetD 中的相互作用可能反映在 DNA 甲基化模式中，特别是在非规范（非-B）DNA 结构中，如 G-四联体（G4s）或 R-环。为了深入了解 MetD 进展的机制，我们专注于两个 MetD 风险基因——葡萄糖激酶（GCK）外显子 7 和跨膜 6 超家族 2（TM6SF2）内含子 2-外显子 3 边界的基因内区域的 DNA 甲基化和功能分析，这些区域具有 G4s 和 R-环的非-B DNA 基序。对嵌套队列研究的 148 份血样进行焦磷酸测序显示，MetD 患者与健康对照组相比，GCK 和 TM6SF2 存在显著的差异甲基化。此外，这些区域在肝癌与癌症基因组图谱（TCGA）的正常组织样本中也存在高度可变和差异甲基化的 CpG。高锰酸盐/S1 核酸酶足迹法与直接接头连接（PDAL-Seq）、天然聚丙烯酰胺 DNA 凝胶电泳和圆二色性（CD）光谱法显示，这些区域形成了 G4 结构，并证明其拓扑结构和稳定性受 DNA 甲基化的影响。包括组蛋白标记、染色质构象捕获数据和荧光素酶报告基因检测在内的详细分析突出了靶区域的细胞类型特异性调节功能。基于我们的分析，我们假设 DNA 甲基化的变化导致拓扑变化，特别是在 GCK 外显子 7 中，从而激活替代调节元件或可能在选择性剪接中发挥作用。我们的分析为 MetD 进展及其与肝癌之间的关联的潜在机制提供了新的视角，揭示了非-B DNA 结构作为重要的关键因素，甚至在早期疾病阶段就发挥作用。
