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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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Immunoprecipitated mdig from mdig-GFP expressing cells can demethylate H3K9me3A. Mass spectrum diagrams showing the mdig-mediated demethylation of H3K9me3. Top panel: incubation of H3K9me3-histone H3 peptide with control IgG; middle panel: incubation of H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 1.5 h; bottom panel: H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 3.0 h.B. Quantification of the demethylation products.
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Figure 4: Immunoprecipitated mdig from mdig-GFP expressing cells can demethylate H3K9me3A. Mass spectrum diagrams showing the mdig-mediated demethylation of H3K9me3. Top panel: incubation of H3K9me3-histone H3 peptide with control IgG; middle panel: incubation of H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 1.5 h; bottom panel: H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 3.0 h.B. Quantification of the demethylation products.

Mentions: As a key regulator and epigenetic marker of heterochromatin, H3K9me3 has been extensively studied for its role in the conformation, maintenance and propagation of heterochromatin. The removal of H3K9me3 by histone demethylases decondenses the heterochromatin structure and de-represses the expression of genes in the heterochromatin region featured with tandemly repeated DNA. Uncertainties remain regarding whether mdig possesses histone demethylase activity based on some cellular and biochemical studies [2,16]. To gain direct evidence showing that mdig may contribute to the demethylation of H3K9me3, we incubated immunoprecipitated mdig from the lysates of mdig-overexpressing cells with a histone H3 peptide containing H3K9me3 for 1.5 and 3 h, respectively. Demethylation activity was then determined by tandem mass spectrometry. As shown in Fig. 4, the incubation of the lysine 9 tri-methylated histone H3 peptide for 1.5 h resulted in the appearance of H3K9me2, H3K9me1, and H3K9me0 peptides (Fig. 4A, middle panel, and Fig. 4B). The concentrations of these peptides with different methylation states were increased further when the time of incubation was increased from 1.5 h to 3 h (Fig. 4A, bottom panel, and Fig. 4B). The quantification of the demethylation activity of the immunoprecipitated mdig protein toward the histone H3 peptide substrate suggested that mdig is able to remove one, two and three methyl groups, with the strongest capability to remove two methyl groups from H3K9me3 (Fig. 4B). To exclude the possibility that such a demethylation was a result of contaminated cellular proteins due to non-specific binding in the immunoprecipitation step, we also performed this demethylation reaction using immunoprecipitated c-Jun, a known nuclear protein, or GAPDH, a cytosolic protein. No demethylation activity was detected when the immunoprecipitated c-Jun or GAPDH was included (data not shown).


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)

Immunoprecipitated mdig from mdig-GFP expressing cells can demethylate H3K9me3A. Mass spectrum diagrams showing the mdig-mediated demethylation of H3K9me3. Top panel: incubation of H3K9me3-histone H3 peptide with control IgG; middle panel: incubation of H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 1.5 h; bottom panel: H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 3.0 h.B. Quantification of the demethylation products.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immunoprecipitated mdig from mdig-GFP expressing cells can demethylate H3K9me3A. Mass spectrum diagrams showing the mdig-mediated demethylation of H3K9me3. Top panel: incubation of H3K9me3-histone H3 peptide with control IgG; middle panel: incubation of H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 1.5 h; bottom panel: H3K9me3-histone H3 peptide with immunoprecipitated mdig protein for 3.0 h.B. Quantification of the demethylation products.
Mentions: As a key regulator and epigenetic marker of heterochromatin, H3K9me3 has been extensively studied for its role in the conformation, maintenance and propagation of heterochromatin. The removal of H3K9me3 by histone demethylases decondenses the heterochromatin structure and de-represses the expression of genes in the heterochromatin region featured with tandemly repeated DNA. Uncertainties remain regarding whether mdig possesses histone demethylase activity based on some cellular and biochemical studies [2,16]. To gain direct evidence showing that mdig may contribute to the demethylation of H3K9me3, we incubated immunoprecipitated mdig from the lysates of mdig-overexpressing cells with a histone H3 peptide containing H3K9me3 for 1.5 and 3 h, respectively. Demethylation activity was then determined by tandem mass spectrometry. As shown in Fig. 4, the incubation of the lysine 9 tri-methylated histone H3 peptide for 1.5 h resulted in the appearance of H3K9me2, H3K9me1, and H3K9me0 peptides (Fig. 4A, middle panel, and Fig. 4B). The concentrations of these peptides with different methylation states were increased further when the time of incubation was increased from 1.5 h to 3 h (Fig. 4A, bottom panel, and Fig. 4B). The quantification of the demethylation activity of the immunoprecipitated mdig protein toward the histone H3 peptide substrate suggested that mdig is able to remove one, two and three methyl groups, with the strongest capability to remove two methyl groups from H3K9me3 (Fig. 4B). To exclude the possibility that such a demethylation was a result of contaminated cellular proteins due to non-specific binding in the immunoprecipitation step, we also performed this demethylation reaction using immunoprecipitated c-Jun, a known nuclear protein, or GAPDH, a cytosolic protein. No demethylation activity was detected when the immunoprecipitated c-Jun or GAPDH was included (data not shown).

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