Dataset Information

Epigenetic regulation and functional characterization of microRNA-142 in mesenchymal cells.

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.

SUBMITTER: Skarn M

PROVIDER: S-EPMC3827369 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Epigenetic regulation and functional characterization of microRNA-142 in mesenchymal cells.

Skårn Magne M Barøy Tale T Stratford Eva Wessel EW Myklebost Ola O

PloS one 20131113 11

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 withi ...[more]

PMID: 24236112

Similar Datasets

Project description:The D1 dopamine receptor subtype is expressed in the brain, kidney and lymphocytes. D1 receptor function has been extensively studied and the receptor has been shown to modulate a wide range of physiological functions and behaviors. The expression of D1 receptor is known to change during development, disease states and chronic treatment; however, the molecular mechanisms that mediate the changes in D1 receptor expression under these circumstances are not well understood. While previous studies have identified extracellular factors and signaling mechanisms regulating the transcription of D1 receptor gene, very little is known about other regulatory mechanisms that modulate the expression of the D1 receptor gene. Here we report that the D1 receptor is post-transcriptionally regulated during postnatal mouse brain development and in the mouse CAD catecholaminergic neuronal cell line. We demonstrate that this post-transcriptional regulation is mediated by a molecular mechanism involving noncoding RNA. We show that the 1277 bp 3'untranslated region of D1 receptor mRNA is necessary and sufficient for mediating the post-transcriptional regulation. Using deletion and site-directed mutagenesis approaches, we show that the D1 receptor post-transcriptional regulation is specifically mediated by microRNA miR-142-3p interacting with a single consensus binding site in the 1277 bp 3'untranslated region of D1 receptor mRNA. Inhibiting endogenous miR-142-3p in CAD cells increased endogenous D1 receptor protein expression levels. The increase in D1 receptor protein levels was biologically significant as it resulted in enhanced D1 receptor-mediated signaling, determined by measuring the activation of both, adenylate cyclase and, the dopamine- and cAMP-regulated phosphoprotein, DARPP-32. We also show that there is an inverse correlation between miR-142-3p levels and D1 receptor protein expression in the mouse brain during postnatal development. This is the first study to demonstrate that the post-transcriptional regulation of D1 receptor expression is mediated by microRNA-induced translational suppression.

Project description:BackgroundLineage commitment of mesenchymal stem cells (MSCs) to cardiac differentiation is controlled by transcription factors that are regulated by epigenetic events, mainly histone deacetylation and promoter DNA methylation. Here, we studied the differentiation of human Wharton's jelly MSCs (WJMSCs) into the cardiomyocyte lineage via epigenetic manipulations.MethodsWe introduced these changes using inhibitors of DNA methyl transferase and histone deacetylase, DC301, DC302, and DC303, in various combinations. We characterized for cardiogenic differentiation by assessing the expression of cardiac-specific markers by immunolocalization, quantitative RT-PCR, and flow cytometry. Cardiac functional studies were performed by FURA2AM staining and Greiss assay. The role of Wnt signaling during cardiac differentiation was analyzed by quantitative RT-PCR. In-vivo studies were performed in a doxorubicin-induced cardiotoxic mouse model by injecting cardiac progenitor cells. Promoter methylation status of the cardiac transcription factor Nkx2.5 and the Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), after cardiac differentiation was studied by bisulfite sequencing.ResultsBy induction with DC301 and DC302, WJMSCs differentiated into cardiomyocyte-like structures with an upregulation of Wnt antagonists, sFRP3 and sFRP4, and Dickkopf (Dkk)1 and Dkk3. The cardiac function enhancer, vinculin, and DDX20, a DEAD-box RNA helicase, were also upregulated in differentiated cardiomyocytes. Additionally, bisulfite sequencing revealed, for the first time in cardiogenesis, that sFRP4 is activated by promoter CpG island demethylation. In vivo, these MSC-derived cardiac progenitors could not only successfully engraft to the site of cardiac injury in mice with doxorubicin-induced cardiac injury, but also form functional cardiomyocytes and restore cardiac function.ConclusionThe present study unveils a link between Wnt inhibition and epigenetic modification to initiate cardiac differentiation, which could enhance the efficacy of stem cell therapy for ischemic heart disorders.

Project description:Epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transitions occur in the development of human tumorigenesis and are part of the natural history of the process to adapt to the changing microenvironment. In this setting, the miR-200 family is recognized as a master regulator of the epithelial phenotype by targeting ZEB1 and ZEB2, two important transcriptional repressors of the cell adherence (E-cadherin) and polarity (CRB3 and LGL2) genes. Recently, the putative DNA methylation associated inactivation of various miR-200 members has been described in cancer. Herein, we show that the miR-200ba429 and miR-200c141 transcripts undergo a dynamic epigenetic regulation linked to EMT or MET phenotypes in tumor progression. The 5'-CpG islands of both miR-200 loci were found unmethylated and coupled to the expression of the corresponding miRNAs in human cancer cell lines with epithelial features, such as low levels of ZEB1/ZEB2 and high expression of E-cadherin, CRB3 and LGL2, while CpG island hypermethylation-associated silencing was observed in transformed cells with mesenchymal characteristics. The recovery of miR-200ba429 and miR-200c141 expression by stable transfection in the hypermethylated cells restored the epithelial markers and inhibited migration in cell culture and tumoral growth and metastasis formation in nude mice. We also discovered, using both cell culture and animal models, that the miR-200 epigenetic silencing is not an static and fixed process but it can be shifted to hypermethylated or unmethylated 5'-CpG island status corresponding to the EMT and MET phenotypes, respectively. In fact, careful laser microdissection in human primary colorectal tumorigenesis unveiled that in normal colon mucosa crypts (epithelia) and stroma (mesenchyma) already are unmethylated and methylated at these loci, respectively; and that the colorectal tumors undergo selective miR-200 hypermethylation of their epithelial component. These findings indicate that the epigenetic silencing plasticity of the miR-200 family contributes to the evolving and adapting phenotypes of human tumors.

Dataset Information

Epigenetic regulation and functional characterization of microRNA-142 in mesenchymal cells.

Publications

Epigenetic regulation and functional characterization of microRNA-142 in mesenchymal cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets