Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
Department of Developmental Biology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
J Am Soc Nephrol. 2023 Oct 1;34(10):1672-1686. doi: 10.1681/ASN.0000000000000197. Epub 2023 Jul 25.
HNF4 genes promote proximal tubule differentiation in mice, but their function in human nephrogenesis is not fully defined. This study uses human pluripotent stem cell (PSC)-derived kidney organoids as a model to investigate HNF4A and HNF4G functions. The loss of HNF4A , but not HNF4G , impaired reabsorption-related molecule expression and microvilli formation in human proximal tubules. Cleavage under targets and release using nuclease (CUT&RUN) sequencing and CRISPR-mediated transcriptional activation (CRISPRa) further confirm that HNF4A directly regulates its target genes. Human kidney organoids provide a good model for studying transcriptional regulation in human kidney development.
The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated.
We generated HNF4A -knock out (KO), HNF4G -KO, and HNF4A/4G -double KO human pluripotent stem cell lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A-binding sites genome-wide by cleavage under targets and release using nuclease sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation.
Organoids lacking HNF4A , but not HNF4G , showed reduced expression of transport-related, endocytosis-related, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. Cleavage under targets and release using nuclease revealed that HNF4A primarily bound promoters and enhancers of genes that were downregulated in HNF4A -KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPR-mediated transcriptional activation drove increased expression of selected target genes during kidney organoid differentiation.
This study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.
HNF4 基因在小鼠中促进近端肾小管分化，但它们在人类肾发生中的功能尚未完全确定。本研究使用人多能干细胞 (PSC) 衍生的肾脏类器官作为模型，研究 HNF4A 和 HNF4G 的功能。HNF4A 的缺失，但不是 HNF4G 的缺失，损害了人类近端肾小管中与重吸收相关的分子表达和微绒毛形成。靶标下切割和释放使用核酸酶 (CUT&RUN) 测序和 CRISPR 介导的转录激活 (CRISPRa) 进一步证实 HNF4A 直接调节其靶基因。人类肾脏类器官为研究人类肾脏发育中的转录调控提供了良好的模型。
近端小管在电解质稳态中起着重要作用。先前的研究表明，HNF4A 调节与重吸收相关的基因，并在小鼠肾脏发育过程中促进近端小管分化。然而，HNF4 家族基因在人类肾发生中的功能和基因调控机制尚未得到研究。
我们生成了 HNF4A 敲除 (KO)、HNF4G KO 和 HNF4A/4G 双 KO 人多能干细胞系，将其各自分化为肾脏类器官，并通过免疫荧光分析、电子显微镜和 RNA-seq 进行分析。我们通过在人成年肾脏和肾脏类器官衍生的近端肾小管细胞中进行靶标下切割和释放使用核酸酶测序，对 HNF4A 结合位点进行全基因组探测。CRISPR 介导的转录激活验证了 HNF4A 和 HNF4G 在肾脏类器官分化过程中近端小管中的功能。
缺乏 HNF4A 的类器官，而不是 HNF4G，表现出转运相关、内吞相关和刷状缘相关基因的表达减少，以及类器官近端肾小管顶端腔中刷状缘结构紊乱。靶标下切割和释放使用核酸酶揭示了 HNF4A 主要结合在 HNF4A-KO 下调基因的启动子和增强子上，表明直接调节。CRISPR 介导的转录激活诱导的 HNF4A 或 HNF4G 的表达驱动了在肾脏类器官分化过程中选定靶基因的表达增加。
本研究揭示了 HNF4A 和 HNF4G 在人类近端小管分化过程中的调控机制。该实验策略可更广泛地应用于研究人类肾脏发育中的转录调控。
