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 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.

Keywords: Cis-regulatory elements; Epididymis epithelium; HNF1; Transcriptional network.

Figure 1. Regional HNF1α and HNF1β expression in HEE cells

(A) Western blot of caput, corpus and cauda cell lysates shows that both HNF1α (90 kDa) and HNF1β (70 kDa) are most abundant in caput cells. (B) Cellular fractionation shows HNF1α and HNF1β are mainly in nuclei of caput cells.

Figure 2. HNF1 occupancy genome-wide in primary human caput HEE cells

(A) Percentage of HNF1 peaks within intergenic, intronic, promoter and other sites (including exons, 5′UTRs and 3′UTRs). (B) Caput DNase-seq tag counts measured from the center of the HNF1 peaks. (C) De novo motif analysis within HNF1 ChIP-seq peaks. (D) Gene ontology analysis by DAVID on the nearest genes to each HNF1 peak. (E) ChIP-qPCR validation of HNF1 binding to 5 sites identified by ChIP-seq, with values relative to IgG control.

Figure 3. HNF1-depletion reveals its role in epididymis epithelial function

(A) Efficacy of siRNA-mediated depletion of HNF1 in caput HEE cells shown by western blot of cell lysates probed with an antibody detecting HNF1α (90 kDa) and HNF1β (70 kDa). Negative control siRNA (NC). (B) Densitometry of western blot in A). (C) RT-qPCR validation of differentially expressed genes in HNF1-siRNA-depleted and non-targeting siRNA treated cells. Data are normalized to β-2 microglobulin (mean ± SD, n = 3) in comparison with negative control siRNA (NC, black bars, HNF1 α/β siRNA white bar). *P < 0.01, **P < 0.001 and ***P < 0.0001 versus NC siRNA-transfected cells.

Figure 4. Identification of novel cis-regulatory elements coinciding with HNF1 ChIP-seq peaks in HNF1-regulated genes

Caput HEE open chromatin identified by DNase-seq are shown above each gene, together with HNF1 ChIP-seq peaks. Arrows denote open chromatin site coinciding with HNF1 ChIP-seq peaks (A) SLC4A2 locus, promoter site. (B) SLC4A3 locus, intergenic site DHS 20 kb downstream. (C) SLC4A4 locus, sites in the promoter and within introns 6 and 20. (D) PKHD1 locus, sites in the promoter and intron 5.

Figure 5. Effect of HNF1 knockdown on intracellular pH (pHi) of caput HEE cells

(A) pHi calibration of caput HEE cells. (B) Effect of HNF1 depletion on pHi. Efficacy of siRNA-mediated depletion of HNF1 in BCECF-loaded cells (B, inset). (C) Effect of DIDS on pHi. BCECF-loaded cells were incubated in Ringers buffer containing 130mM NaCl for 5min. Repetitive dual fluorescence readings (excitation 444nm and 488nm, emission 535nm) were recorded and converted to pHi Representative traces are shown. Each point represents the mean ± S.E.M. (n = 3). *P < 0.05, versus NC siRNA-transfected cells.