Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity

Aims/hypothesis: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. Methods: Single cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. Results: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "bHI" cells and were diminished in db/db mice. "Hub" cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretation: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Comparative gene expression profiling analysis of RNA-seq data for NNAT-positive vs NNAT-negative primary mouse beta cells.研究目的与假设：胰腺胰岛中的β细胞(Beta cells)属于异质性群体，不同细胞亚群对胰岛素分泌的整体调控发挥独特作用。其中包含一类高连接“枢纽”细胞，其在细胞间钙离子(Ca²+)波的传播过程中发挥关键作用。人类β细胞中同样存在功能亚群，且2型糖尿病患者体内可见该亚群的分布发生改变。目前，学界对β细胞层级结构建立的分子机制尚不清楚。表观基因组层面的改变正是其中一种潜在调控途径，本研究聚焦于印迹基因神经元蛋白(neuronatin, Nnat)展开探究，该基因对正常胰岛素的合成与分泌至关重要。 方法：本研究采用R语言环境下运行的Seurat 4.0与ClusterProfiler工具，对单细胞RNA测序(scRNA-seq)数据集进行分析。我们构建了在Nnat增强子/启动子区域调控下表达增强型绿色荧光蛋白(eGFP)的转基因小鼠，用于胰岛β细胞的荧光激活细胞(FAC)分选，并分别通过亚硫酸氢盐测序与RNA测序开展CpG甲基化与转录组的后续分析。我们使用自胰腺祖细胞阶段起缺失从头甲基转移酶(de novo methyltransferase)DNMT3A(DNMT3A)的基因工程小鼠，探究启动子甲基化的调控机制。蛋白质组学分析采用亲和纯化质谱技术完成；钙离子动态变化则通过Cal-520与Cal-590的快速共聚焦成像进行检测。胰岛素分泌水平采用均相时间分辨荧光成像技术进行测定。 结果：Nnat mRNA在特定β细胞亚群中呈差异性表达，且该表达模式依赖于发育阶段与DNA甲基化(DNMT3A)状态。对胚胎数据集的拟时间分析显示，在胚胎发育过程中，Nnat阳性与阴性亚群很早就得以形成。成人胰腺胰岛中，NNAT的表达同样局限于特定β细胞亚群，且该亚群在整个成年生命周期中均得以维持。成年阶段的NNAT阳性β细胞同样具有独特的转录组特征，属于专门负责胰岛素生成的亚群，与近期报道的"bHI"细胞特征相似，且在db/db小鼠体内该亚群的丰度显著降低。NNAT阳性亚群中“枢纽”细胞的丰度更低，这与该功能特化的表观遗传调控机制相符。 结论与阐释：本研究结果表明，Nnat位点的差异性DNA甲基化是发育过程中β细胞异质性建立的全新调控方式。因此我们提出假设：该位点的甲基化改变可能会导致β细胞层级结构与细胞连接性丧失，进而可能在部分糖尿病类型中引发胰岛素分泌功能缺陷。本研究针对NNAT阳性与NNAT阴性的原代小鼠β细胞的RNA测序数据，开展了比较基因表达谱分析。