Genome-wide analysis of PDX1 target genes in human pancreatic progenitors [expression profiling]

Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing β-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions included important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B and MEIS1, which were activated during the differentiation process as revealed by the active mono-acetylated chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand for the Notch receptor, DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult β-cell function. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provides stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a β-cell developmental program. We performed Affymetrix microarray analysis on XM001 cells at the pluripotency and PP stages

研究背景（Objective）：
编码同源盒转录因子（homeobox transcription factor, TF）PDX1的基因发生纯合功能丧失性突变时，可引发胰腺发育不全；而杂合突变则可导致青少年发病型糖尿病4型（Maturity-Onset Diabetes of the Young 4, MODY4）。尽管在临床前模型中，学界已对PDX1在胰岛素分泌β细胞发育与稳态维持中的功能开展了充分研究，但该转录因子如何通过调控下游转录程序控制人类胰腺发育，目前仍不明晰。此外，诸多研究均报道了单核苷酸多态性（single nucleotide polymorphisms, SNPs）与2型糖尿病（type 2 diabetes mellitus, T2DM）之间的关联，且已有研究证实胰岛增强子在T2DM相关SNPs中富集。截至目前，尚不清楚携带T2DM相关SNPs的基因组区域是否在胰腺祖细胞阶段结合PDX1并处于激活状态。

研究方法（Methods）：
本研究构建了一株可高效分化为人类胰腺祖细胞（pancreatic progenitors, PPs）的新型诱导多能干细胞（induced pluripotent stem cell, iPSC）系。此外，本研究通过PDX1与H3K27ac染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq），鉴定了PDX1的转录靶基因以及激活的增强子与启动子区域。为探究PDX1在发育阶段与成年阶段的功能差异，本研究对比了胰腺祖细胞与成人胰岛中的PDX1结合谱。同时，本研究结合ChIP-seq与全基因组关联研究（genome-wide association study, GWAS）荟萃分析数据，在PDX1结合位点与激活染色质区域中鉴定出了T2DM相关SNPs。

研究结果（Results）：
针对PDX1的ChIP-seq分析显示，在诱导多能干细胞分化得到的胰腺祖细胞中，共鉴定到8088个PDX1结合区域，对应5664个靶基因。PDX1的靶区域包含多个关键胰腺转录因子，例如PDX1自身、RFX6、HNF1B与MEIS1；通过活性单乙酰化染色质标记H3K27ac与mRNA表达谱分析可见，这些基因在分化过程中被激活，提示自身调控反馈环路可维持PDX1的表达，并启动胰腺转录因子程序。值得注意的是，本研究鉴定出数个此前尚未在人类中报道过的PDX1靶基因，包括在小鼠中参与纤毛发生与内分泌分化的RFX3，以及在内分泌诱导与尖端-躯干模式形成中发挥重要作用的Notch受体配体DLL1。对比胰腺祖细胞与成人胰岛的PDX1结合谱，本研究鉴定出多组阶段特异性靶基因，这些基因分别与胰腺早期发育及成人β细胞功能相关。此外，本研究发现诱导多能干细胞分化得到的胰腺祖细胞的激活染色质区域中，T2DM相关SNPs存在显著富集。其中两个SNPs位于TCF7L2与HNF1B的内含子区域内的PDX1结合位点中。这两个基因均为内分泌诱导的关键转录调控因子，其顺式调控区域的突变可增加糖尿病患病风险。

研究结论（Conclusions）：
本研究的数据揭示了干细胞体外分化为胰腺祖细胞过程中PDX1的阶段特异性靶基因，这可为鉴定糖尿病易感通路与分子靶标提供重要参考。此外，本研究证实T2DM相关SNPs在胰腺祖细胞阶段的激活染色质区域中富集，提示2型糖尿病的易感性可能源于β细胞发育程序的执行缺陷。本研究还对处于多能性阶段与胰腺祖细胞阶段的XM001细胞进行了Affymetrix基因芯片分析。