Abstract
The transcripts encoded by the microRNA mir-142 gene are highly active in hematopoietic cells, but expressed at low levels in many other cell types. Treatment with the demethylating agent 5-Aza-2′-deoxycytidine increased both the 1,636 nucleotide primary transcript and mature miR-142-5p/3p in mesenchymal cells, indicating that mir-142 is epigenetically repressed by DNA methylation. The transcription start site was determined to be located 1,205 base pairs upstream of the precursor sequence within a highly conserved CpG island. In addition, a second CpG island overlapped with the precursor. A TATA-box, several promoter-proximal elements and enrichment of conserved transcription factor binding sites within the first 100 base pairs upstream of the transcription start site, suggests that this region represents the core/proximal mir-142 promoter. Moreover, both CpG islands were heavily methylated in mesenchymal cells, having low levels of miR-142-5p/3p, and unmethylated in hematopoietic cells where both miRNAs were abundantly expressed. We show that treatment with 5-Aza-2′-deoxycytidine significantly reduced the DNA methylation of the upstream CpG island, which led to increased expression, and that in vitro DNA methylation of the upstream region of the mir-142 precursor repressed its transcriptional activity. When overexpressed, miR-142-5p/3p reduced proliferation of cells with epigenetic silencing of endogenous mir-142. This finding is interesting as miR-142-5p/3p have been reported to be deregulated in tumors of mesenchymal origin. We provide the first experimental evidence that transcription of mir-142 is directly repressed by DNA methylation. In addition, we discovered that the antisense strand of mir-142 might act as a precursor for functional mature antisense miRNAs. Thus, our study expands the current knowledge about the regulation of mir-142 and function of miR-142-5p/3p, and adds novel insight into the rapidly increasing field of microRNA regulation.
Highlights
IntroductionMicroRNAs (miRNAs) are short endogenous non-coding RNAs (ncRNAs) that inhibit the expression of target genes at the posttranscriptional level [1]
MicroRNAs are short endogenous non-coding RNAs that inhibit the expression of target genes at the posttranscriptional level [1]
We suggest that transcription of mir-142 in mesenchymal cells is repressed by DNA methylation of an upstream conserved promoter-associated CpG islands (CGIs), and that the cell type-specific expression of mir-142 could be predominantly determined by methylation
Summary
MicroRNAs (miRNAs) are short endogenous non-coding RNAs (ncRNAs) that inhibit the expression of target genes at the posttranscriptional level [1]. MiRNAs are estimated to regulate 30–60% of protein-coding genes [2,3]. Among 939 human miRNAs analyzed by Sato et al, 31% were intergenic (i.e. located between annotated protein-coding genes), whereas the remaining were intragenic; localized within either protein-coding or ncRNA transcripts in either sense (44%), antisense (13%) or both orientations (12%) [5]. The intergenic miRNAs are independent transcription units, with their own regulatory elements [4], whereas intragenic miRNAs are co-transcribed with their host genes [6], or have their own promoters [7,8]. Transcription and processing of the strand antisense to an annotated miRNA have been reported in several studies from Drosophila [9,10,11] and mammals [12,13]
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