Integrative Cistromic and Transcriptomic Analyses Identify CREB Target Genes in Cystic Renal Epithelial Cells
Integrative Cistromic and Transcriptomic Analyses Identify CREB Target Genes in Cystic Renal Epithelial Cells
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顺反组学和转录组学综合分析鉴定囊性肾上皮细胞中的 CREB 靶基因
DOI：
10.1681/asn.2021010101
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发表时间：
2021-10-01
影响因子：
13.6
通讯作者：
Chen, Yupeng
中科院分区：
文献类型：
--
作者：
Liu, Zhiheng;Liu, Yunjing;Chen, Yupeng
背景转录因子结合位点的全基因组定位对于确定转录因子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特定细胞类型的数量有限，在体内确定转铁蛋白的结合位置一直是一个挑战。CAMP反应元件结合蛋白(CREB)在常染色体显性遗传性多囊肾病(ADPKD)中被磷酸化和过度激活。我们以CREB为例，分析Tf结合的基因组位点，并利用较少的特异肾脏细胞来确定其靶基因。方法以正常和ADPKD小鼠肾小管上皮细胞为材料，进行靶下切割和核酸酶释放(Cut&Run)分析。用ADPKD小鼠模型研究了666-15对CREB的药理抑制作用和A-CREB的遗传抑制作用。结果对囊性上皮细胞中磷酸化CREB(p-CREB)的全基因组分布进行切割和扫描分析表明，p-CREB结合与活化组蛋白修饰(H3K4me3和H3K27ac)相关。整合分析结合切割和RNA测序揭示了CREB的直接靶点，包括参与核糖体生物发生和蛋白质合成的基因。结论CREB通过激活核糖体生物发生基因促进囊变发生。Cut&Run结合转录分析，能够从少量特定的肾脏细胞中询问TF结合并识别直接的TF靶标。
Background 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.Methods 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.Results 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.Conclusions 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.