Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
Department of Preventative Medicine, Division of Bioinformatics, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
PLoS Genet. 2018 Jan 29;14(1):e1007181. doi: 10.1371/journal.pgen.1007181. eCollection 2018 Jan.
Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.
肾祖细胞数量决定了肾单位的数量；肾单位数量减少与肾脏疾病的发生有关。几个转录调节因子，包括 Six2、Wt1、Osr1、Sall1、Eya1、Pax2 和 Hox11 同源物，对于肾祖细胞的特化和/或维持是必需的。然而，人们对这些因子的调控交集知之甚少。在这里，我们绘制了 Six2、Hoxd11、Osr1 和 Wt1 的肾祖细胞特异性转录网络。我们确定了与祖细胞程序密切相关的基因周围的 373 个多因素相关的“调控热点”。为了研究它们的功能意义，我们删除了 Six2 和 Wnt4 的“热点”增强子元件。去除 Six2 的远端增强子导致 Six2 表达减少约 40%。当与 Six2 缺失等位基因结合时，祖细胞表现出肾祖细胞的过早耗竭。Wnt4 增强子的缺失导致肾小泡中 Wnt4 表达的显著减少和肾脏轻度发育不良，这种表型在与 Wnt4 缺失突变结合时也得到增强。为了探索支持适当靶基因表达的调控景观，我们进行了 CTCF ChIP-seq 以鉴定绝缘子结合区域。这样的一个假定边界位于 Six2 和 Six3 基因座之间。通过对辐射诱导的 Brachyrrhine (Br) 突变等位基因进行深度测序，获得了该边界的功能重要性的证据。我们发现 Six2/Six3 基因座周围的 Six2 位点发生了反转，将 Six2 从其远端增强子调控中去除，但放置在 Six3 增强子元件旁边，这些元件支持 Six3 在正常表达 Six3 的晶状体中异位表达 Six2。Six3 现在被预测受 Six2 远端增强子的控制。与这一观点一致，我们观察到 Six3 在肾祖细胞中异位表达。4C-seq 支持了野生型和 Br/+ 小鼠肾脏中 Six2 远端增强子相互作用的模型。总之，这些数据扩展了我们对调控基因组和调控景观的认识，这些基因组和调控景观是哺乳动物肾发生的基础。
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