2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Institute of Urology, Tianjin Medical University, Tianjin, China.
Department of Toxicology and Sanitary Chemistry, School of Public Health, Tianjin Medical University, Tianjin, China.
J Am Soc Nephrol. 2021 Oct;32(10):2529-2541. doi: 10.1681/ASN.2021010101. Epub 2021 Jun 23.
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 . 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 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.
全基因组范围内转录因子（TF）结合位点的作图对于鉴定肾脏发育和疾病中 TF 的直接靶基因至关重要。然而，由于肾脏细胞的复杂性和特定细胞类型数量有限，确定 TF 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）中发生磷酸化和过度活跃。我们以 CREB 为例，使用数量有限的特定肾脏细胞来分析基因组上 TF 结合的位点，并鉴定其靶基因。
使用正常和 ADPKD 小鼠肾脏中的凝集素（DBA）阳性管状上皮细胞进行靶标切割和释放（CUT&RUN）测定。使用 666-15 对 CREB 进行药理学抑制和使用 A-CREB 进行基因抑制，在 ADPKD 小鼠模型中进行。
CUT&RUN 用于分析全基因组范围内磷酸化 CREB（p-CREB）的分布，表明 p-CREB 结合与囊性上皮细胞中活性组蛋白修饰（H3K4me3 和 H3K27ac）相关。CUT&RUN 与 RNA-seq 的综合分析揭示了 CREB 的直接靶基因，包括参与核糖体生物发生和蛋白质合成的基因。CREB 的药理学和基因抑制抑制了 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 与转录组分析相结合，能够从少量特定肾脏细胞中探究 TF 结合并鉴定直接 TF 靶标。
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