Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo, 162-8655, Japan.
Department of Preventive Environment and Nutrition, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Diabetologia. 2018 Dec;61(12):2608-2620. doi: 10.1007/s00125-018-4735-7. Epub 2018 Oct 3.
AIMS/HYPOTHESIS: Epigenetic regulation of gene expression has been implicated in the pathogenesis of obesity and type 2 diabetes. However, detailed information, such as key transcription factors in pancreatic beta cells that mediate environmental effects, is not yet available.
To analyse genome-wide cis-regulatory profiles and transcriptome of pancreatic islets derived from a diet-induced obesity (DIO) mouse model, we conducted chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) of histone H3 lysine 27 acetylation (histone H3K27ac) and high-throughput RNA sequencing. Transcription factor-binding motifs enriched in differential H3K27ac regions were examined by de novo motif analysis. For the predicted transcription factors, loss of function experiments were performed by transfecting specific siRNA in INS-1, a rat beta cell line, with and without palmitate treatment. Epigenomic and transcriptional changes of possible target genes were evaluated by ChIP and quantitative RT-PCR.
After long-term feeding with a high-fat diet, C57BL/6J mice were obese and mildly glucose intolerant. Among 39,350 islet cis-regulatory regions, 13,369 and 4610 elements showed increase and decrease in ChIP-Seq signals, respectively, significantly associated with global change in gene expression. Remarkably, increased H3K27ac showed a distinctive genomic localisation, mainly in the proximal-promoter regions, revealing enriched elements for nuclear respiratory factor 1 (NRF1), GA repeat binding protein α (GABPA) and myocyte enhancer factor 2A (MEF2A) by de novo motif analysis, whereas decreased H3K27ac was enriched for v-maf musculoaponeurotic fibrosarcoma oncogene family protein K (MAFK), a known negative regulator of beta cells. By siRNA-mediated knockdown of NRF1, GABPA or MEF2A we found that INS-1 cells exhibited downregulation of fatty acid β-oxidation genes in parallel with decrease in the associated H3K27ac. Furthermore, in line with the epigenome in DIO mice, palmitate treatment caused increase in H3K27ac and induction of β-oxidation genes; these responses were blunted when NRF1, GABPA or MEF2A were suppressed.
CONCLUSIONS/INTERPRETATION: These results suggest novel roles for DNA-binding proteins and fatty acid signalling in obesity-induced epigenomic regulation of beta cell function.
The next-generation sequencing data in the present study were deposited at ArrayExpress. RNA-Seq: Dataset name: ERR2538129 (Control), ERR2538130 (Diet-induced obesity) Repository name and number: E-MTAB-6718 - RNA-Seq of pancreatic islets derived from mice fed a long-term high-fat diet against chow-fed controls. ChIP-Seq: Dataset name: ERR2538131 (Control), ERR2538132 (Diet-induced obesity) Repository name and number: E-MTAB-6719 - H3K27ac ChIP-Seq of pancreatic islets derived from mice fed a long-term high-fat diet (HFD) against chow-fed controls.
目的/假设：基因表达的表观遗传调控与肥胖和 2 型糖尿病的发病机制有关。然而，目前尚缺乏有关介导环境影响的胰腺β细胞关键转录因子的详细信息。
为了分析饮食诱导肥胖（DIO）小鼠模型来源的胰岛的全基因组顺式调控谱和转录组，我们进行了组蛋白 H3 赖氨酸 27 乙酰化（组蛋白 H3K27ac）的染色质免疫沉淀结合高通量测序（ChIP-Seq）和高通量 RNA 测序。通过从头 motif 分析，研究了差异 H3K27ac 区域中富集的转录因子结合基序。对于预测的转录因子，我们通过转染特异性 siRNA，在大鼠β细胞系 INS-1 中进行了功能丧失实验，同时进行了棕榈酸处理。通过 ChIP 和定量 RT-PCR 评估了可能的靶基因的表观基因组和转录组变化。
经过长期高脂肪饮食喂养，C57BL/6J 小鼠肥胖且轻度葡萄糖不耐受。在 39350 个胰岛顺式调控区域中，有 13369 个和 4610 个元件的 ChIP-Seq 信号分别增加和减少，与基因表达的整体变化显著相关。值得注意的是，增加的 H3K27ac 显示出独特的基因组定位，主要位于近端启动子区域，通过从头 motif 分析揭示了富含核呼吸因子 1（NRF1）、GA 重复结合蛋白α（GABPA）和肌细胞增强因子 2A（MEF2A）的富集元件，而减少的 H3K27ac 则富含已知的β细胞负调控因子 v-maf 肌肉骨骼纤维肉瘤癌基因家族蛋白 K（MAFK）。通过 siRNA 介导的 NRF1、GABPA 或 MEF2A 敲低，我们发现 INS-1 细胞中脂肪酸β氧化基因的表达下调，同时与 H3K27ac 的减少相关。此外，与 DIO 小鼠的表观基因组一致，棕榈酸处理导致 H3K27ac 增加和β氧化基因诱导；当抑制 NRF1、GABPA 或 MEF2A 时，这些反应被减弱。
结论/解释：这些结果表明，在肥胖诱导的β细胞功能的表观遗传调控中，DNA 结合蛋白和脂肪酸信号具有新的作用。
本研究中的下一代测序数据已存储在 ArrayExpress 中。RNA-Seq：数据集名称：ERR2538129（对照），ERR2538130（饮食诱导肥胖）存储库名称和编号：E-MTAB-6718 - 来自长期高脂肪饮食喂养的小鼠的胰岛的 RNA-Seq 与对照喂养的小鼠相比。ChIP-Seq：数据集名称：ERR2538131（对照），ERR2538132（饮食诱导肥胖）存储库名称和编号：E-MTAB-6719 - 来自长期高脂肪饮食喂养的小鼠的胰岛的 H3K27ac ChIP-Seq 与对照喂养的小鼠相比。
