Genome-wide mapping of transcription factor (TF) binding sites is essential to identify a TF’s direct target genes in kidney development and diseases. However, due to the cellular complexity of the kidney and limited numbers of a given cell type, it has been challenging to determine the binding sites of a TF in vivo. cAMP response element-binding protein (CREB) is phosphorylated and hyperactive in autosomal dominant polycystic kidney disease (ADPKD). We focus on CREB as an example to profile genomic loci bound by a TF and to identify its target genes using low numbers of specific kidney cells.

Cleavage under targets and release using nuclease (CUT&RUN) assays were performed with Dolichos biflorus agglutinin (DBA)–positive tubular epithelial cells from normal and ADPKD mouse kidneys. Pharmacologic inhibition of CREB with 666-15 and genetic inhibition with A-CREB were undertaken using ADPKD mouse models.

CUT&RUN to profile genome-wide distribution of phosphorylated CREB (p-CREB) indicated correlation of p-CREB binding with active histone modifications (H3K4me3 and H3K27ac) in cystic epithelial cells. Integrative analysis with CUT&RUN and RNA-sequencing revealed CREB direct targets, including genes involved in ribosome biogenesis and protein synthesis. Pharmacologic and genetic inhibition of CREB suppressed cyst growth in ADPKD mouse models.

CREB promotes cystogenesis by activating ribosome biogenesis genes. CUT&RUN, coupled with transcriptomic analysis, enables interrogation of TF binding and identification of direct TF targets from a low number of specific kidney cells.

转录因子 (TF) 结合位点的全基因组图谱对于识别 TF 在肾脏发育和疾病中的直接靶基因至关重要。然而，由于肾脏的细胞复杂性和给定细胞类型的数量有限，确定 TF在体内的结合位点一直具有挑战性。cAMP 反应元件结合蛋白 (CREB) 在常染色体显性多囊肾病 (ADPKD) 中被磷酸化和过度活跃。我们以 CREB ​​为例来分析由 TF 结合的基因组位点，并使用少量特定肾细胞识别其靶基因。

使用来自正常和 ADPKD 小鼠肾脏的Dolichos biflorus凝集素 (DBA) 阳性肾小管上皮细胞进行靶点切割和使用核酸酶 (CUT&RUN) 释放。使用 ADPKD 小鼠模型进行了 666-15 对 CREB ​​的药理学抑制和 A-CREB ​​的遗传抑制。

CUT&RUN 以分析磷酸化 CREB ​​(p-CREB) 的全基因组分布表明囊性上皮细胞中 p-CREB ​​结合与活性组蛋白修饰（H3K4me3 和 H3K27ac）的相关性。CUT&RUN 和 RNA 测序的综合分析揭示了 CREB ​​的直接目标，包括参与核糖体生物发生和蛋白质合成的基因。CREB ​​的药理学和遗传抑制抑制了 ADPKD 小鼠模型中的囊肿生长。

CREB ​​通过激活核糖体生物发生基因促进囊肿发生。CUT&RUN 与转录组学分析相结合，能够询问 TF 结合并从少量特定肾细胞中识别直接 TF 靶标。