Epididymal Region-Specific miRNA Expression and DNA Methylation and Their Roles in Controlling Gene Expression in Rats.

Chen Chu,Mofang Liu,Wubin Ma,Guangyong Zheng,Yonglian Zhang,Yuchuan Zhou,Minjie Ni,Shengsong Xie,Jinsong Zhang,Shuanggang Hu,Yixue Li,Lu Zhou,Chunhua Tang,Joël R Drevet

doi:10.1371/journal.pone.0124450

Abstract

Region-specific gene expression is an intriguing feature of the mammalian epididymis. This unique property is essential for sperm maturation and storage, and it also implicates stringent and multi-level regulations of gene expression. Over the past decade, the androgen-driven activation of epididymal gene transcription has been extensively studied. However, it still remains largely unexplored whether and how other regulatory mechanisms, such as miRNAs and DNA methylation, are involved in controlling regional gene expression in the epididymis. Using microarray-based approaches, we studied the regional miRNA expression and DNA methylation profiles in 4 distinct epididymal regions (initial segment, caput, corpus and cauda) of rats. We found that the miR-200 family members were more expressed in caput, compared with cauda. By GSEA analysis, the differential expression of miR-200 family between caput and cauda was shown to be negatively correlated with their predicted target genes, among which 4 bona fide targets were verified by luciferase reporter assay. Predicted target genes of miR-200 family have enriched functions in anti-apoptosis, cell transportation and development, implying the regional diversity in epididymal functions. On the other hand, we revealed epididymal DNA methylation of 2002 CpG islands and 2771 gene promoters (-3.88-0.97kb), among which 1350 (67.43%) CpG islands and 2095 (75.60%) promoters contained region-specific DNA methylation. We observed significant and distinct functional enrichment in genes with specifically methylated promoters in each epididymal regions, but these DNA methylations did not show significant correlation with repressed gene transcription in the mature epididymis. Conclusively, we investigated the regional miRNA expression and DNA methylation in the rat epididymis and revealed a potential role of miR-200 family in gene expression regulation between caput and cauda. This may contribute to the distinct physiological function in sperm maturation / storage of caput / cauda epididymis.

Highlights

Along the mammalian epididymal tubule, epithelial cells secrete proteins in a region-specific manner, creating an ever-changing luminal environment for sperm maturation and storage [1,2,3,4]
To verify the MeDIP-chip results, we examined the promoter methylation status of Mbp, Uox, Slc7a1, Cdkl1 and Cdx2 by bisulfite sequencing PCR (BSP) because they had different ‘Peak scores’ according to the MeDIP-chip results (S1 Table and S1 Fig), i.e. Mbp had the highest peak score among all genes; Uox, Slc7a1 and Cdkl1 were randomly picked genes with lower peak scores; Cdx2 was a randomly picked gene with a peak score
By BSP validation, we found that the ‘Peak score’ of each gene didn’t correlate with their actual promoter methylation levels, the MeDIP-chip data of the tested genes all agreed with the BSP results (S1 File and S1 Fig)

Summary

Introduction

Along the mammalian epididymal tubule, epithelial cells secrete proteins in a region-specific manner, creating an ever-changing luminal environment for sperm maturation and storage [1,2,3,4]. This feature is attributed to the uniquely fine-tuned spatial gene expression pattern of epididymal epithelial cells [5,6]. DNA methylation and miRNAs modulate target gene activities without altering the DNA sequence and respectively regulate gene expression at transcriptional and post-transcriptional levels. Mammalian miRNAs recognize their target mRNAs by 6–8 nucleotides known as the seed region [13]. miRNAs with sequence similarity in the seed regions often share analogous biological functions and can be clustered as miRNA families [14]

Methods

Results

Conclusion