HNF1 regulates critical processes in the human epididymis epithelium

Abstract

The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1β transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to a non-targeting siRNA. Differentially expressed genes with HNF1 ChIP-seq peaks within 20kb were subject to gene ontology process enrichment analysis. Among the most significant pathways associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment.

Transcriptional networks in the human epididymis

“…). We used this approach to map tissue‐specific transcriptional networks in HEE cells, which were then validated by relevant functional assays (Fossum et al ., , ; Gosalia et al ., ; Browne et al ., ; Yang et al ., , 2018).…”

“…First DNase‐seq (or ATAC‐seq) reveals open (active) chromatin regions in human epididymis epithelial (HEE) cells that likely contain cis ‐regulatory elements coordinating gene expression in these cells. Subsequent in silico analysis identifies transcription factor binding sites (TFBS) that are over‐represented in HEE open chromatin (Bischof et al ., ; Browne et al ., , ). ChIP‐seq using well‐validated antibodies specific for individual TFs identifies their sites of occupancy genome‐wide and determines their target genes (Browne et al ., ; Yang et al ., ).…”

“…Subsequent in silico analysis identifies transcription factor binding sites (TFBS) that are over‐represented in HEE open chromatin (Bischof et al ., ; Browne et al ., , ). ChIP‐seq using well‐validated antibodies specific for individual TFs identifies their sites of occupancy genome‐wide and determines their target genes (Browne et al ., ; Yang et al ., ). Furthermore, the contribution of the TF to the transcriptome can be determined by RNA‐seq following siRNA‐mediated TF depletion, or in the case of ligand‐activated TFs such as hormone receptors, upon their activation (Fig.…”

“…As noted in the tissue analysis, very few transcripts were differentially expressed between the corpus and cauda HEE cells, while there were many DEGs between cells from these regions and from the caput. This demonstrated that caput HEE cells are functionally divergent from those of the corpus and cauda, which have similar transcriptomes (Browne et al ., ,b). Several ion and solute transport‐related processes are predominant in caput epithelial cells.…”

“…These include channels and transporters for potassium, chloride, bicarbonate, and water. Also, highly over‐represented in caput cells are pathways of response to hormone stimulus, and inspection of the genes within these pathways revealed a number of relevant hormone receptor genes including the androgen receptor ( AR ), the estrogen receptor ( ESR ) and the glucagon receptor ( GCGR ) (Browne et al ., , ). The enrichment of the AR in the proximal epididymis is consistent with AR immunostaining of adult human epididymis sections (Ungefroren et al ., ).…”