HNF1 regulates critical processes in the human epididymis epithelium

Highlights

• Open chromatin in caput human epididymis epithelial (HEE) cells predicted a key role for HNF1β.

• HNF1 ChIP-seq identified direct target genes of this transcription factor in HEE cells.

• RNA-seq after HNF1α/β depletion defined their contribution to the HEE transcriptome.

• HNF1 has an important role in regulating epithelial water and solute transport in caput HEE cells.

• Intracellular pH values show HNF1 contributes to the control of epididymis luminal environment.

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 non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes 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.

Introduction

The luminal environment in the epididymis is established and maintained by cellular processes in the epithelial layer lining the duct. These functions provide a milieu that is critical for sperm maturation, protection and storage, and hence male fertility. Many genes encode proteins that are required for the normal properties of the luminal fluid, which include ionic concentration, pH, and protein/peptide composition. For example, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), cAMP-activated chloride channel, which is highly expressed in the male genital duct epithelium, are associated with male infertility (Harris and Coleman, 1989; Pollard et al., 1991, Chillon et al., 1995, Jarvi et al., 1995). The network of transcription factors that coordinate expression of important epithelial transport proteins in the human epididymis is not fully characterized, though several mouse models provide clues. For example, infertility of male mice that lack the Foxi1 transcription factor is attributed to misregulation of its target genes encoding the H+-ATPase proton pump, carbonic anhydrase II and the chloride/bicarbonate transporter pendrin (SLC26A4), all of which contribute to luminal fluid composition (Blomqvist et al., 2006). Similarly, infertility in Estrogen Receptor (ESR1)-null mice is attributed to loss of regulation of a key target gene, sodium/hydrogen exchanger 3 (NHE3), in efferent ductules (Zhou et al., 2001). However, many other transcription factors that are pivotal to normal epididymis epithelium function remain to be characterized. We identified hepatocyte nuclear factor 1 (HNF1) as a key epididymis epithelial transcription factor by bioinformatic analysis of genome-wide open chromatin in immature epididymis epithelial cells. The HNF1 motif was over-represented in these cells (Harris and Coleman, 1989, Bischof et al., 2013) and we show here that it is also over-represented in adult human caput epididymis epithelial (HEE) cells (Leir et al., 2015). HNF1α and HNF1β recognize the same DNA consensus sequence, which they occupy as homodimers or heterodimers. Both factors share a high degree of homology in their DNA binding and dimerization (N-terminus) regions but have a more divergent C-terminal transactivation domain. HNF1β is essential for the generation of a functional male reproductive tract in mice (Lokmane et al., 2010), and genital tract abnormalities are evident in humans with recessive mutations in HNF1β (Lindner et al., 1999, Edghill et al., 2006). Though the functions of HNF1α and HNF1β are well studied in epithelia from several other tissues (liver, kidney, intestine, and pancreas), reviewed in Clissold et al. (2015) their role in the adult epididymis epithelium remains unexplored. The human epididymis has 3 functionally distinct regions: the head (caput), body (corpus) and tail (cauda) with different transcriptomes (Zhang et al., 2006, Thimon et al., 2007, Dube et al., 2007, Browne et al., 2015). The caput and cauda luminal environments are probably the most relevant for sperm maturation (Schoysman and Bedford, 1986, Dacheux and Dacheux, 2014). Here we investigate the role of HNF1 in regulating gene expression and important biological processes in the caput epididymis epithelium using primary adult HEE cells (Leir et al., 2015). Following siRNA-mediated depletion of HNF1α and HNF1β we determined the contribution of these factors to the caput HEE cell transcriptome. Next, we integrated these data with genome-wide occupancy of HNF1 in caput epithelial cells, identified by ChIP-seq. Together these datasets reveal multiple novel HNF1-target genes and processes of direct relevance to epididymis function and sperm maturation.