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Methyltransferase Set7/9 maintains transcription and euchromatin structure at islet-enriched genes.

Deering TG, Ogihara T, Trace AP, Maier B, Mirmira RG - Diabetes (2008)

Bottom Line: Within the pancreas, Set7/9 protein shows striking specificity for islet cells, including alpha- and beta-cells, as well as occasional cells within ducts.Consistent with these findings, the Set7/9 gene promoter contained an islet-specific enhancer located between -5,768 and -6,030 base pairs (relative to the transcriptional start site) that exhibited Pdx1-responsive activation in beta-cells.These changes in transcription were accompanied by loss of dimethylated H3 Lys4 and RNA polymerase II recruitment, particularly at the Ins1/2 and Glut2 genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.

ABSTRACT

Objective: The activation of beta-cell genes, particularly of those encoding preproinsulin, requires an appropriate euchromatin (or "open") DNA template characterized by hypermethylation of Lys4 of histone H3. We hypothesized that this modification is maintained in islet beta-cells by the action of the histone methyltransferase Set7/9.

Research design and methods: To identify the role of Set7/9, we characterized its expression pattern and gene regulation and studied its function using RNA interference in both cell lines and primary mouse islets.

Results: Within the pancreas, Set7/9 protein shows striking specificity for islet cells, including alpha- and beta-cells, as well as occasional cells within ducts. Consistent with these findings, the Set7/9 gene promoter contained an islet-specific enhancer located between -5,768 and -6,030 base pairs (relative to the transcriptional start site) that exhibited Pdx1-responsive activation in beta-cells. To study Set7/9 function, we depleted insulinoma cells and primary mouse islets of Set7/9 protein using siRNA. Following siRNA treatment, we observed striking repression of genes involved in glucose-stimulated insulin secretion, including Ins1/2, Glut2, and MafA. These changes in transcription were accompanied by loss of dimethylated H3 Lys4 and RNA polymerase II recruitment, particularly at the Ins1/2 and Glut2 genes. Consistent with these data, depletion of Set7/9 in islets led to defects in glucose-stimulated Ca(2+) mobilization and insulin secretion.

Conclusions: We conclude that Set7/9 is required for normal beta-cell function, likely through the maintenance of euchromatin structure at genes necessary for glucose-stimulated insulin secretion.

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Related in: MedlinePlus

Set7/9 is necessary for the maintenance of dimethylated H3 Lys4 at specific genes in βTC3 cells. βTC3 cells were transfected with the siRNAs indicated and subjected to ChIP using either normal rabbit serum (−Ab) or antibodies to methylated histones. Recovery of the gene fragments was assessed by real-time PCR. Data are reported as recovery of the indicated gene (Ins1/2 or Slc2a2) following ChIP as a percent of the input levels of the gene before ChIP. A: Percent recovery of dimethylated H3 Lys4 at the Ins1/2 promoter and coding regions. B: Percent recovery of momomethylated H3 Lys4 at the Ins1/2 promoter and coding regions. C: Percent recovery of dimethylated H3 Lys4 at the Slc2a2 proximal promoter region. D: Percent recovery of dimethylated H3 Lys4 at the Pdx1 proximal promoter region. Data represent the average of at least three independent ChIP assays from at least three independent siRNA transfections. *Statistically different (P < 0.05) from data of siControl transfections. The amplified region of the genes (in bp) relative to the transcriptional start site is indicated in parentheses below each gene. ▪, siControl; □, siSet97; , siSet98.
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f6: Set7/9 is necessary for the maintenance of dimethylated H3 Lys4 at specific genes in βTC3 cells. βTC3 cells were transfected with the siRNAs indicated and subjected to ChIP using either normal rabbit serum (−Ab) or antibodies to methylated histones. Recovery of the gene fragments was assessed by real-time PCR. Data are reported as recovery of the indicated gene (Ins1/2 or Slc2a2) following ChIP as a percent of the input levels of the gene before ChIP. A: Percent recovery of dimethylated H3 Lys4 at the Ins1/2 promoter and coding regions. B: Percent recovery of momomethylated H3 Lys4 at the Ins1/2 promoter and coding regions. C: Percent recovery of dimethylated H3 Lys4 at the Slc2a2 proximal promoter region. D: Percent recovery of dimethylated H3 Lys4 at the Pdx1 proximal promoter region. Data represent the average of at least three independent ChIP assays from at least three independent siRNA transfections. *Statistically different (P < 0.05) from data of siControl transfections. The amplified region of the genes (in bp) relative to the transcriptional start site is indicated in parentheses below each gene. ▪, siControl; □, siSet97; , siSet98.

Mentions: In prior ChIP studies, we demonstrated that Set7/9 occupies the Ins1/2 genes in βTC3 cells (10). This observation, coupled with the finding that the Ins1/2 gene is hypermethylated at H3 Lys4 (10,11), suggested to us that Set7/9 may be directly responsible for the maintenance of this euchromatin histone modification at this gene. We therefore wondered whether the reduction in Ins1/2 gene activity observed upon knockdown of Set7/9 in this study could be secondary to loss of H3-Lys4 methylation. As shown in Fig. 6A, depletion of Set7/9 in βTC3 cells using either of two siRNAs (siSet97 or siSet98) led to 50–80% reductions in dimethylated H3 Lys4 in the Ins1/2 promoter region as determined by ChIP assay but did not affect dimethylated H3 Lys4 in the coding region of the gene. Importantly, we did not observe any changes to monomethylated H3 Lys4 in either the promoter or coding regions of the Ins1/2 gene (Fig. 6B), suggesting that Set7/9 is associated with a very specific dimethylation effect.


Methyltransferase Set7/9 maintains transcription and euchromatin structure at islet-enriched genes.

Deering TG, Ogihara T, Trace AP, Maier B, Mirmira RG - Diabetes (2008)

Set7/9 is necessary for the maintenance of dimethylated H3 Lys4 at specific genes in βTC3 cells. βTC3 cells were transfected with the siRNAs indicated and subjected to ChIP using either normal rabbit serum (−Ab) or antibodies to methylated histones. Recovery of the gene fragments was assessed by real-time PCR. Data are reported as recovery of the indicated gene (Ins1/2 or Slc2a2) following ChIP as a percent of the input levels of the gene before ChIP. A: Percent recovery of dimethylated H3 Lys4 at the Ins1/2 promoter and coding regions. B: Percent recovery of momomethylated H3 Lys4 at the Ins1/2 promoter and coding regions. C: Percent recovery of dimethylated H3 Lys4 at the Slc2a2 proximal promoter region. D: Percent recovery of dimethylated H3 Lys4 at the Pdx1 proximal promoter region. Data represent the average of at least three independent ChIP assays from at least three independent siRNA transfections. *Statistically different (P < 0.05) from data of siControl transfections. The amplified region of the genes (in bp) relative to the transcriptional start site is indicated in parentheses below each gene. ▪, siControl; □, siSet97; , siSet98.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Set7/9 is necessary for the maintenance of dimethylated H3 Lys4 at specific genes in βTC3 cells. βTC3 cells were transfected with the siRNAs indicated and subjected to ChIP using either normal rabbit serum (−Ab) or antibodies to methylated histones. Recovery of the gene fragments was assessed by real-time PCR. Data are reported as recovery of the indicated gene (Ins1/2 or Slc2a2) following ChIP as a percent of the input levels of the gene before ChIP. A: Percent recovery of dimethylated H3 Lys4 at the Ins1/2 promoter and coding regions. B: Percent recovery of momomethylated H3 Lys4 at the Ins1/2 promoter and coding regions. C: Percent recovery of dimethylated H3 Lys4 at the Slc2a2 proximal promoter region. D: Percent recovery of dimethylated H3 Lys4 at the Pdx1 proximal promoter region. Data represent the average of at least three independent ChIP assays from at least three independent siRNA transfections. *Statistically different (P < 0.05) from data of siControl transfections. The amplified region of the genes (in bp) relative to the transcriptional start site is indicated in parentheses below each gene. ▪, siControl; □, siSet97; , siSet98.
Mentions: In prior ChIP studies, we demonstrated that Set7/9 occupies the Ins1/2 genes in βTC3 cells (10). This observation, coupled with the finding that the Ins1/2 gene is hypermethylated at H3 Lys4 (10,11), suggested to us that Set7/9 may be directly responsible for the maintenance of this euchromatin histone modification at this gene. We therefore wondered whether the reduction in Ins1/2 gene activity observed upon knockdown of Set7/9 in this study could be secondary to loss of H3-Lys4 methylation. As shown in Fig. 6A, depletion of Set7/9 in βTC3 cells using either of two siRNAs (siSet97 or siSet98) led to 50–80% reductions in dimethylated H3 Lys4 in the Ins1/2 promoter region as determined by ChIP assay but did not affect dimethylated H3 Lys4 in the coding region of the gene. Importantly, we did not observe any changes to monomethylated H3 Lys4 in either the promoter or coding regions of the Ins1/2 gene (Fig. 6B), suggesting that Set7/9 is associated with a very specific dimethylation effect.

Bottom Line: Within the pancreas, Set7/9 protein shows striking specificity for islet cells, including alpha- and beta-cells, as well as occasional cells within ducts.Consistent with these findings, the Set7/9 gene promoter contained an islet-specific enhancer located between -5,768 and -6,030 base pairs (relative to the transcriptional start site) that exhibited Pdx1-responsive activation in beta-cells.These changes in transcription were accompanied by loss of dimethylated H3 Lys4 and RNA polymerase II recruitment, particularly at the Ins1/2 and Glut2 genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.

ABSTRACT

Objective: The activation of beta-cell genes, particularly of those encoding preproinsulin, requires an appropriate euchromatin (or "open") DNA template characterized by hypermethylation of Lys4 of histone H3. We hypothesized that this modification is maintained in islet beta-cells by the action of the histone methyltransferase Set7/9.

Research design and methods: To identify the role of Set7/9, we characterized its expression pattern and gene regulation and studied its function using RNA interference in both cell lines and primary mouse islets.

Results: Within the pancreas, Set7/9 protein shows striking specificity for islet cells, including alpha- and beta-cells, as well as occasional cells within ducts. Consistent with these findings, the Set7/9 gene promoter contained an islet-specific enhancer located between -5,768 and -6,030 base pairs (relative to the transcriptional start site) that exhibited Pdx1-responsive activation in beta-cells. To study Set7/9 function, we depleted insulinoma cells and primary mouse islets of Set7/9 protein using siRNA. Following siRNA treatment, we observed striking repression of genes involved in glucose-stimulated insulin secretion, including Ins1/2, Glut2, and MafA. These changes in transcription were accompanied by loss of dimethylated H3 Lys4 and RNA polymerase II recruitment, particularly at the Ins1/2 and Glut2 genes. Consistent with these data, depletion of Set7/9 in islets led to defects in glucose-stimulated Ca(2+) mobilization and insulin secretion.

Conclusions: We conclude that Set7/9 is required for normal beta-cell function, likely through the maintenance of euchromatin structure at genes necessary for glucose-stimulated insulin secretion.

Show MeSH
Related in: MedlinePlus