目的:缺乏bHLH转录因子(TF)Neurog3的小鼠不形成胰岛细胞,包括分泌胰岛素的β细胞,缺乏会导致糖尿病。在人类中,NEUROG3纯合突变表现为新生儿或儿童糖尿病。尽管在胰岛细胞发育中起着关键作用,NEUROG3直接调控的基因程序和下游基因程序的精确功能仍然难以捉摸。因此,我们在人类诱导多能干细胞(hiPSC)来源的内分泌祖细胞中绘制了全基因组NEUROG3的占有率,并确定了NEUROG3对相关基因的依赖性,以揭示直接的靶标.
方法:我们产生了一个新的hiPSC细胞系(NEUROG3-HA-P2A-Venus),其中NEUROG3被HA标记并融合到自切割荧光VENUS报告基因。我们使用CUT&RUN技术绘制了与该hiPSC系不同的胰腺内分泌祖细胞(PEP)中NEUROG3的占有率和表观遗传标记。我们将NEUROG3占有率数据与PEP中的染色质状态和基因表达以及它们的NEUROG3依赖性进行了整合。此外,我们调查了NEUROG3在PEP阶段是否与2型糖尿病(T2DM)相关变异体结合.
结果:CUT&RUN显示共有863个NEUROG3结合位点分配给1263个独特基因。在启动子以及在PEP活性增强子中经常重叠的远处顺式调节元件中发现了NEUROG3占用。从头基序分析定义了NEUROG3共有结合基序,并提示FOXA或RFX转录因子对NEUROG3靶基因的潜在共调节。我们发现22%的基因在神经元3-/-PEP中下调,在NEUROG3-Venus阳性内分泌细胞中富集的基因中有10%被NEUROG3结合,因此可能被直接调节。NEUROG3与138个转录因子基因结合,一些在胰岛细胞发育或功能中起重要作用,如NEUROD1,PAX4,NKX2-2,SOX4,MLXIPL,LMX1B,RFX3和NEUROG3本身,和许多其他未知的胰岛功能。出乎意料的是,我们发现NEUROG3靶向对β细胞胰岛素分泌至关重要的基因(例如,GCK,ABCC8/KCNJ11,CACNA1A,CHGA,SCG2、SLC30A8和PCSK1)。因此,分析NEUROG3的占有率表明,NEUROG3的瞬时表达不仅促进了未定型胰腺祖细胞的胰岛命运,但也可以启动对β细胞功能至关重要的内分泌程序。最后,我们在NEUROG3结合区域内确定了8个T2DM风险SNP.
结论:在PEP中定位NEUROG3基因组占有率发现出乎意料的广泛,直接控制内分泌基因,提出了关于这种主要调节因子如何控制胰岛和β细胞分化的新假设。
OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets.
METHODS: We generated a novel hiPSC line (
NEUROG3-HA-P2A-Venus) where
NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their
NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage.
RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of
NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in
NEUROG3-/- PEPs, and 10% of genes enriched in
NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of
NEUROG3 occupancy suggests that the transient expression of
NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions.
CONCLUSIONS: Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.