Integrative Cistromic and Transcriptomic Analyses Identify CREB Target Genes in Cystic Renal Epithelial Cells

Significance Statement Transcription factors (TFs) play crucial roles in kidney development and diseases by recognizing specific DNA sequences to control gene expression programs. The kidney’s cellular heterogeneity poses substantial challenges to identifying the genomic binding sites and direct target genes of TFs in vivo . We apply the cleavage under targets and release using nuclease (CUT&RUN) technique, together with transcriptomic analysis, to identify cAMP response element-binding protein (CREB) target genes in cystic epithelial cells of autosomal dominant polycystic kidney disease (ADPKD) in a mouse model. CREB binds to and activates ribosomal biogenesis genes, and inhibition of CREB retards cyst growth in the ADPKD models. CUT&RUN is a powerful method for genome-scale profiling and identifying direct targets of TFs from small numbers of specific kidney cells. 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.