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Mdig de-represses H19 large intergenic non-coding RNA (lincRNA) by down-regulating H3K9me3 and heterochromatin.

Chen B, Yu M, Chang Q, Lu Y, Thakur C, Ma D, Yi Z, Chen F - Oncotarget (2013)

Bottom Line: Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression.Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3.Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy, Wayne State University, 259 Mack Avenue, Detroit, MI, USA.

ABSTRACT
Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

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Mdig enhances the expression of genes in the heterochromatin regionA. Quantitative real-time PCR showing that mdig overexpression increases the expression of H19, IGF2 and macrosatellite X56, which are normally repressed by the heterochromatin conformation. B. Quantitative real-time PCR showing that mdig upregulates the expression of c-Myc and Jhdm3a. C. Quantitative real-time PCR showing that silencing mdig by shRNA suppressed the expression of H19. D. Chromatin immunoprecipitation (ChIP) assays showing that mdig overexpression reduces the enrichment of H3K9me3 in the promoter region of the H19 gene (left panel). Silencing mdig by shRNA increased the enrichment of H3K9me3 in the promoter (middle panel) and the ICR region (right panel) of the H19 gene.
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Figure 3: Mdig enhances the expression of genes in the heterochromatin regionA. Quantitative real-time PCR showing that mdig overexpression increases the expression of H19, IGF2 and macrosatellite X56, which are normally repressed by the heterochromatin conformation. B. Quantitative real-time PCR showing that mdig upregulates the expression of c-Myc and Jhdm3a. C. Quantitative real-time PCR showing that silencing mdig by shRNA suppressed the expression of H19. D. Chromatin immunoprecipitation (ChIP) assays showing that mdig overexpression reduces the enrichment of H3K9me3 in the promoter region of the H19 gene (left panel). Silencing mdig by shRNA increased the enrichment of H3K9me3 in the promoter (middle panel) and the ICR region (right panel) of the H19 gene.

Mentions: Our immunoblotting data showed that mdig only exhibited a very marginal effect on the total H3K9me3 level in the cells in which mdig is overexpressed or silenced by siRNA or shRNA (Fig. 2). One possibility of such a weak effect may be a result of the limited activity of mdig on the global level of H3K9me3 which is distributed in a wide range of chromatin types, including constitutive heterochromatin, facultative heterochromatin, repressive and non-repressive euchromatin, etc. To determine the potential influence of mdig on a limited scale of epigenetic regulation, we focused on the effect of mdig on the transcription of genes that are known to be silenced in the heterochromatin regions, such as satellite and imprinting loci. H19 and IGF2 are paternally and maternally imprinted genes, respectively, due to the highly repetitive DNA elements between these two gene loci and the developmental formation of a highly condensed heterochromatin structure in their gene loci. Quantitative RT-PCR experiments showed that the transcript levels of H19 and IGF2 were elevated 9.98-fold and 1.7-fold, respectively, in the cells that stably overexpressed mdig (Fig. 3A). In addition to H19 and IGF2, we also investigated the level of a long non-coding transcript from the macrosatellite X56 that is packaged in facultative heterochromatin characterized by H3K9me3 and CpG DNA hypermethylation at the active X chromosome [34]. The overexpression of mdig induces a 2.96-fold increase of macrosatellite X56 transcript levels (Fig. 3A, right panel). Furthermore, 2.8-fold and 3.4-fold increases of c-Myc and Jhdm3a expression were observed in the mdig-overexpressing cells relative to the cells expressing a control vector (Fig. 3B). The increased expression of c-Myc may be important in mediating the growth-promoting feature of the mdig protein, whereas elevated Jhdm3a, a known H3K9me3 demethylase, may contribute to the demethylation of H3K9me3 and possibly H3K36me3 in the mdig-expressing cells [35].


Mdig de-represses H19 large intergenic non-coding RNA (lincRNA) by down-regulating H3K9me3 and heterochromatin.

Chen B, Yu M, Chang Q, Lu Y, Thakur C, Ma D, Yi Z, Chen F - Oncotarget (2013)

Mdig enhances the expression of genes in the heterochromatin regionA. Quantitative real-time PCR showing that mdig overexpression increases the expression of H19, IGF2 and macrosatellite X56, which are normally repressed by the heterochromatin conformation. B. Quantitative real-time PCR showing that mdig upregulates the expression of c-Myc and Jhdm3a. C. Quantitative real-time PCR showing that silencing mdig by shRNA suppressed the expression of H19. D. Chromatin immunoprecipitation (ChIP) assays showing that mdig overexpression reduces the enrichment of H3K9me3 in the promoter region of the H19 gene (left panel). Silencing mdig by shRNA increased the enrichment of H3K9me3 in the promoter (middle panel) and the ICR region (right panel) of the H19 gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Mdig enhances the expression of genes in the heterochromatin regionA. Quantitative real-time PCR showing that mdig overexpression increases the expression of H19, IGF2 and macrosatellite X56, which are normally repressed by the heterochromatin conformation. B. Quantitative real-time PCR showing that mdig upregulates the expression of c-Myc and Jhdm3a. C. Quantitative real-time PCR showing that silencing mdig by shRNA suppressed the expression of H19. D. Chromatin immunoprecipitation (ChIP) assays showing that mdig overexpression reduces the enrichment of H3K9me3 in the promoter region of the H19 gene (left panel). Silencing mdig by shRNA increased the enrichment of H3K9me3 in the promoter (middle panel) and the ICR region (right panel) of the H19 gene.
Mentions: Our immunoblotting data showed that mdig only exhibited a very marginal effect on the total H3K9me3 level in the cells in which mdig is overexpressed or silenced by siRNA or shRNA (Fig. 2). One possibility of such a weak effect may be a result of the limited activity of mdig on the global level of H3K9me3 which is distributed in a wide range of chromatin types, including constitutive heterochromatin, facultative heterochromatin, repressive and non-repressive euchromatin, etc. To determine the potential influence of mdig on a limited scale of epigenetic regulation, we focused on the effect of mdig on the transcription of genes that are known to be silenced in the heterochromatin regions, such as satellite and imprinting loci. H19 and IGF2 are paternally and maternally imprinted genes, respectively, due to the highly repetitive DNA elements between these two gene loci and the developmental formation of a highly condensed heterochromatin structure in their gene loci. Quantitative RT-PCR experiments showed that the transcript levels of H19 and IGF2 were elevated 9.98-fold and 1.7-fold, respectively, in the cells that stably overexpressed mdig (Fig. 3A). In addition to H19 and IGF2, we also investigated the level of a long non-coding transcript from the macrosatellite X56 that is packaged in facultative heterochromatin characterized by H3K9me3 and CpG DNA hypermethylation at the active X chromosome [34]. The overexpression of mdig induces a 2.96-fold increase of macrosatellite X56 transcript levels (Fig. 3A, right panel). Furthermore, 2.8-fold and 3.4-fold increases of c-Myc and Jhdm3a expression were observed in the mdig-overexpressing cells relative to the cells expressing a control vector (Fig. 3B). The increased expression of c-Myc may be important in mediating the growth-promoting feature of the mdig protein, whereas elevated Jhdm3a, a known H3K9me3 demethylase, may contribute to the demethylation of H3K9me3 and possibly H3K36me3 in the mdig-expressing cells [35].

Bottom Line: Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression.Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3.Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy, Wayne State University, 259 Mack Avenue, Detroit, MI, USA.

ABSTRACT
Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

Show MeSH
Related in: MedlinePlus