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Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

ABSTRACT: The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human epididymis epithelial (HEE) cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in silico analysis identified transcription factor-binding sites (TFBS) that were over-represented in the HEE open chromatin, including the motif for paired box 2 (PAX2). PAX2 is a critical transcriptional regulator of urogenital tract development, which has been well studied in the kidney but is unexplored in the epididymis. Due to the limited lifespan of primary HEE cells in culture, we investigated the role of PAX2 in an immortalized HEE cell line (REP). First, REP cells were evaluated by DNase I digestion followed by high-throughput sequencing and the PAX2-binding motif was again identified as an over-represented TFBS within intergenic open chromatin, though on fewer chromosomes than in the primary HEE cells. To identify PAX2-target genes in REP cells, RNA-seq analysis was performed after siRNA-mediated depletion of PAX2 and compared with that with a non-targeting siRNA. In response to PAX2-repression, 3135 transcripts were differentially expressed (1333 up-regulated and 1802 down-regulated). Novel PAX2 targets included multiple genes encoding proteins with predicted functions in the epididymis epithelium.

SUBMITTER: Browne JA

PROVIDER: S-EPMC4235575 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

Browne James A JA Yang Rui R Song Lingyun L Crawford Gregory E GE Leir Shih-Hsing SH Harris Ann A

Molecular human reproduction 20140901 12

The epithelium lining the epididymis in the male reproductive tract maintains a luminal environment that promotes sperm cell maturation. This process is dependent on the coordinated expression of many genes that encode proteins with a role in epithelial transport. We previously generated genome-wide maps of open chromatin in primary human epididymis epithelial (HEE) cells to identify potential regulatory elements controlling coordinated gene expression in the epididymis epithelium. Subsequent in ...[more]

PMID: 25180270

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Project description:BACKGROUND:The epithelial lining of the human epididymis is critical for sperm maturation. This process requires distinct specialized functions in the head, body, and tail of the duct. These region-specific properties are maintained by distinct gene expression profiles which are governed by transcription factor networks, non-coding RNAs, and other factors. MATERIALS AND METHODS:We used genome-wide protocols including DNase-seq, RNA-seq and ChIP-seq to characterize open (active) chromatin, the transcriptome and occupancy of specific transcription factors (TFs) respectively, in caput, corpus, and cauda segments of adult human epididymis tissue and primary human epididymis epithelial (HEE) cell cultures derived from them. RNA-seq following TF depletion or activation, combined with gene ontology analysis also determined TF targets. RESULTS:Among regional differentially expressed transcripts were epithelial-selective transcription factors (TFs), microRNAs, and antiviral response genes. Caput-enriched TFs included hepatocyte nuclear factor 1 (HNF1) and the androgen receptor (AR), both of which were also predicted to occupy cis-regulatory elements identified as open chromatin in HEE cells. HNF1 targets were identified genome-wide using ChIP-seq, in HEE cells. Next, siRNA-mediated depletion of HNF1 revealed a pivotal role for this TF in coordinating epithelial water and solute transport in caput epithelium. The importance of AR in HEE cells was shown by AR ChIP-seq, and by RNA-seq after synthetic androgen (R1881) treatment. AR has a distinct transcriptional program in the HEE cells and likely recruits different co-factors (RUNX1 and CEBP?) in comparison to those used in prostate epithelium. DISCUSSION AND CONCLUSION:Our data identify many transcription factors that regulate the development and differentiation of HEE cells. Moreover, a comparison between immature and adult HEE cells showed key TFs in the transition to fully differentiated function of this epithelium. These data may help identify new targets to treat male infertility and have the potential to open new avenues for male contraception.

Dataset Information

Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

Publications

Open chromatin mapping identifies transcriptional networks regulating human epididymis epithelial function.

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