Identification of direct transcriptional targets of Nfatc2 that promote ß-cell proliferation in human islets (ncRNA-seq)

The transcription factor Nfatc2 is a potent ß-cell mitogen in mouse and human islets, however, the direct genomic targets that mediate the mitogenic effects have not been identified. We expressed a constitutively active form of Nfatc2, and the closely related Nfatc1, in human islets and identified ~5,600 differentially expressed genes. Nfatc2 binding sites in human islets were identified via ChIP-seq, yielding ~8,600 high-confidence peaks. By integrating gene expression changes induced by Nfatc2 with genomic binding sites for Nfatc2, we identified ~2,200 direct transcriptional targets of Nfatc2 in human islets. Direct targets that were induced by Nfatc2 were enriched for transcripts that regulate the cell cycle, and for motifs associated with the transcription factor FoxP1. We showed that islets from an endocrine-specific FoxP1, FoxP2 and FoxP4 triple-knockout mouse are less responsive to Nfatc2-induced ß-cell proliferation, suggesting the FoxP family may work in concert with Nfatc2 to regulate ß-cell proliferation. Nfatc2 induced ß-cell proliferation in both mouse and human islets, whereas Nfatc1 does so only in human islets. Exploiting this species difference in Nfatc1-responsiveness, we identified ~250 direct transcriptional targets with similar transcriptional profiles. This gene set strongly enriches for cell cycle-associated transcripts, and includes the nuclear hormone receptor, Nr4a1. We show that deletion of Nr4a1 greatly reduces the capacity of Nfatc2 to induce ß-cell proliferation, suggesting that much of the effect on Nfatc2 occurs through its induction of Nr4a1. Integration of ncRNA profiling, chromatin accessibility and Nfatc2 binding sites enabled us to identify key Nfatc2-dependent enhancer loci that mediate enhanced ß-cell proliferation. Overall design: Whole-islet RNA was collected from 5 separate human donors 48 hours after adenovirus (Ad)-mediated expression of constitutively active (ca) Nfatc2 or Ad-GFP. ncRNAs were enriched using RiboZero purification and oligo-dT depletion.

转录因子（transcription factor）Nfatc2是小鼠和人类胰岛中一种强效的β细胞有丝分裂原，然而介导其促有丝分裂效应的直接基因组靶标尚未被鉴定。我们在人类胰岛中过表达Nfatc2及其近缘家族成员Nfatc1的组成型激活形式，鉴定出约5600个差异表达基因。通过染色质免疫沉淀测序（ChIP-seq），我们鉴定了人类胰岛中Nfatc2的结合位点，获得约8600个高置信度结合峰。将Nfatc2诱导的基因表达变化与Nfatc2的基因组结合位点进行整合分析，我们在人类胰岛中鉴定出约2200个Nfatc2的直接转录靶标。受Nfatc2诱导的直接靶标富集于调控细胞周期的转录本，以及与转录因子FoxP1相关的DNA结合基序。我们证实，内分泌组织特异性FoxP1、FoxP2和FoxP4三基因敲除小鼠的胰岛对Nfatc2诱导的β细胞增殖反应性显著降低，这提示FoxP家族可能与Nfatc2协同调控β细胞增殖。Nfatc2可在小鼠和人类胰岛中诱导β细胞增殖，而Nfatc1仅在人类胰岛中发挥该效应。利用Nfatc1反应性的物种差异，我们鉴定出约250个具有相似转录谱的直接转录靶标。该基因集显著富集细胞周期相关转录本，其中包含核激素受体Nr4a1。我们证实，敲除Nr4a1可大幅降低Nfatc2诱导β细胞增殖的能力，这表明Nfatc2的大部分调控效应是通过其诱导Nr4a1的表达实现的。整合非编码RNA（ncRNA，non-coding RNA）表达谱、染色质开放性数据与Nfatc2结合位点，我们得以鉴定出关键的Nfatc2依赖性增强子位点，这些位点介导了β细胞增殖的增强。整体实验设计：分别向5名独立人类供体的胰岛中通过腺病毒（adenovirus, Ad）介导组成型激活型（ca）Nfatc2或Ad-GFP的过表达，48小时后收集全胰岛RNA。非编码RNA通过RiboZero纯化与寡聚dT去除法进行富集。