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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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Marginal effect of mdig on total H3K9me3A & B. Western blotting detection for the mdig protein and the histone H3 methylation status in the cells stably transfected with control shRNA or mdig shRNA. C. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells transfected with a control siRNA or mdig siRNA. D. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells stably expressing a control vector or mdig. E. Immunofluorescent staining of H3K9me3 in the cells stably expressing an RFP-tagged control shRNA or an RFP-tagged mdig shRNA. Nuclei were staining by DAPI.
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Figure 2: Marginal effect of mdig on total H3K9me3A & B. Western blotting detection for the mdig protein and the histone H3 methylation status in the cells stably transfected with control shRNA or mdig shRNA. C. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells transfected with a control siRNA or mdig siRNA. D. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells stably expressing a control vector or mdig. E. Immunofluorescent staining of H3K9me3 in the cells stably expressing an RFP-tagged control shRNA or an RFP-tagged mdig shRNA. Nuclei were staining by DAPI.

Mentions: The mdig protein contains a conversed JmjC domain, the signature motif of the histone demethylase family of proteins. Although previous studies showed a reduced level of H3K9me3 in the BEAS-2B cells transfected with mdig, direct evidence regarding whether mdig is a histone demethylase is still lacking. In the stably transfected A549 cells that express mdig shRNA that silences mdig, we noted about a 50 to 60% reduction of mdig protein. However, only a very marginal increase of H3K9me3 and H3K9me1 was observed in these cells (Fig. 2A). No notable effect of mdig on the methylation states of H3K27 was observed (Fig. 2B). To exclude the possibility that the shRNA had a poor silencing effect on mdig in the stably transfected cells, we also used siRNA to silence mdig in A549 cells. Although the siRNA showed much improved efficacy on silencing mdig (Fig. 2C), again, only a marginal increase of H3K9me3 was detected.


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)

Marginal effect of mdig on total H3K9me3A & B. Western blotting detection for the mdig protein and the histone H3 methylation status in the cells stably transfected with control shRNA or mdig shRNA. C. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells transfected with a control siRNA or mdig siRNA. D. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells stably expressing a control vector or mdig. E. Immunofluorescent staining of H3K9me3 in the cells stably expressing an RFP-tagged control shRNA or an RFP-tagged mdig shRNA. Nuclei were staining by DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824531&req=5

Figure 2: Marginal effect of mdig on total H3K9me3A & B. Western blotting detection for the mdig protein and the histone H3 methylation status in the cells stably transfected with control shRNA or mdig shRNA. C. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells transfected with a control siRNA or mdig siRNA. D. Western blotting detection of the mdig protein and the histone H3 methylation status in the cells stably expressing a control vector or mdig. E. Immunofluorescent staining of H3K9me3 in the cells stably expressing an RFP-tagged control shRNA or an RFP-tagged mdig shRNA. Nuclei were staining by DAPI.
Mentions: The mdig protein contains a conversed JmjC domain, the signature motif of the histone demethylase family of proteins. Although previous studies showed a reduced level of H3K9me3 in the BEAS-2B cells transfected with mdig, direct evidence regarding whether mdig is a histone demethylase is still lacking. In the stably transfected A549 cells that express mdig shRNA that silences mdig, we noted about a 50 to 60% reduction of mdig protein. However, only a very marginal increase of H3K9me3 and H3K9me1 was observed in these cells (Fig. 2A). No notable effect of mdig on the methylation states of H3K27 was observed (Fig. 2B). To exclude the possibility that the shRNA had a poor silencing effect on mdig in the stably transfected cells, we also used siRNA to silence mdig in A549 cells. Although the siRNA showed much improved efficacy on silencing mdig (Fig. 2C), again, only a marginal increase of H3K9me3 was detected.

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