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The histone demethylase Jhdm1a regulates hepatic gluconeogenesis.

Pan D, Mao C, Zou T, Yao AY, Cooper MP, Boyartchuk V, Wang YX - PLoS Genet. (2012)

Bottom Line: In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level.Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity.This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus.

View Article: PubMed Central - PubMed

Affiliation: Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.

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Suppression of gluconeogenic gene expression by Jhdm1a requires its demethylation activity.(A) (Left panel) Jhdm1a constructs were tagged with HA and transfected into Hela cells to ensure they express stable proteins. Co-transfected Flag-PPARδ serves as transfection and loading control. (Right, three panels) Jhdm1a constructs were lentivirally expressed in HepG2 cells. Data were shown from one representative of five experiments with similar results. (B) Lentiviral expression of Jhdm1b does not suppress gluconeogenic gene expression. (C) Jhdm1a constructs were lentivirally expressed in rat hepatic FAO cells and glucose production was measured. Data were from triplicates. **, P<0.01.
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pgen-1002761-g002: Suppression of gluconeogenic gene expression by Jhdm1a requires its demethylation activity.(A) (Left panel) Jhdm1a constructs were tagged with HA and transfected into Hela cells to ensure they express stable proteins. Co-transfected Flag-PPARδ serves as transfection and loading control. (Right, three panels) Jhdm1a constructs were lentivirally expressed in HepG2 cells. Data were shown from one representative of five experiments with similar results. (B) Lentiviral expression of Jhdm1b does not suppress gluconeogenic gene expression. (C) Jhdm1a constructs were lentivirally expressed in rat hepatic FAO cells and glucose production was measured. Data were from triplicates. **, P<0.01.

Mentions: Given that knockdown of Jhdm1a elevates gluconeogenic gene expression, we examined whether an opposite effect could be observed in cells expressing Jhdm1a. We stably expressed Jhdm1a via lentivirus in liver cells and found that this expression decreased both basal and hormonal-stimulated levels of PEPCK and G6Pase mRNA (Figure 2A and Figure S3). Interestingly, and in agreement with the knockdown data (Figure 1B), ectopic expression of demethylase Jhdm1b, which is closely related to Jhdm1a, did not inhibit gluconeogenic gene expression (Figure 2B and Figure S4). To determine the domains in Jhdm1a that are required for its suppressive function, we generated a series of Jhdm1a mutants. We first confirmed that these mutants were capable of producing stable proteins at a similar level, as judged by plasmid transfection in Hela cells (Figure 2A). We then expressed the mutants in HepG2 cells through lentivirus with a similar, low infection efficiency. Deletion of the JmjC domain or the CXXC Zinc finger domain abolished the suppression on PEPCK and G6Pase expression, whereas mutant lacking either the PHD domain or the F-box and Leucine-rich repeats remained fully functional (Figure 2A and Figure S3). Note that these Jhdm1a mutants were expressed at similar mRNA levels as their wild-type counterpart. The JmjC domain harbors the histone demethylation activity. Consistent with the effect of the JmjC deletion mutant, a demethylation-dead point mutant (H212A) [20] of Jhdm1a was no longer able to suppress PEPCK and G6Pase expression (Figure 2A and Figure S3). We next determined the effect of Jhdm1a on glucose production in vitro. We found that ectopic expression of wild type Jhdm1a, but not the demethylation defective mutants, inhibited glucose production in rat hepatoma FAO cells (Figure 2C). Taken together, these results demonstrate that both the demethylation activity and the CXXC Zinc finger domain of Jhdm1a are required for its negative modulation of gluconeogenic gene expression.


The histone demethylase Jhdm1a regulates hepatic gluconeogenesis.

Pan D, Mao C, Zou T, Yao AY, Cooper MP, Boyartchuk V, Wang YX - PLoS Genet. (2012)

Suppression of gluconeogenic gene expression by Jhdm1a requires its demethylation activity.(A) (Left panel) Jhdm1a constructs were tagged with HA and transfected into Hela cells to ensure they express stable proteins. Co-transfected Flag-PPARδ serves as transfection and loading control. (Right, three panels) Jhdm1a constructs were lentivirally expressed in HepG2 cells. Data were shown from one representative of five experiments with similar results. (B) Lentiviral expression of Jhdm1b does not suppress gluconeogenic gene expression. (C) Jhdm1a constructs were lentivirally expressed in rat hepatic FAO cells and glucose production was measured. Data were from triplicates. **, P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375226&req=5

pgen-1002761-g002: Suppression of gluconeogenic gene expression by Jhdm1a requires its demethylation activity.(A) (Left panel) Jhdm1a constructs were tagged with HA and transfected into Hela cells to ensure they express stable proteins. Co-transfected Flag-PPARδ serves as transfection and loading control. (Right, three panels) Jhdm1a constructs were lentivirally expressed in HepG2 cells. Data were shown from one representative of five experiments with similar results. (B) Lentiviral expression of Jhdm1b does not suppress gluconeogenic gene expression. (C) Jhdm1a constructs were lentivirally expressed in rat hepatic FAO cells and glucose production was measured. Data were from triplicates. **, P<0.01.
Mentions: Given that knockdown of Jhdm1a elevates gluconeogenic gene expression, we examined whether an opposite effect could be observed in cells expressing Jhdm1a. We stably expressed Jhdm1a via lentivirus in liver cells and found that this expression decreased both basal and hormonal-stimulated levels of PEPCK and G6Pase mRNA (Figure 2A and Figure S3). Interestingly, and in agreement with the knockdown data (Figure 1B), ectopic expression of demethylase Jhdm1b, which is closely related to Jhdm1a, did not inhibit gluconeogenic gene expression (Figure 2B and Figure S4). To determine the domains in Jhdm1a that are required for its suppressive function, we generated a series of Jhdm1a mutants. We first confirmed that these mutants were capable of producing stable proteins at a similar level, as judged by plasmid transfection in Hela cells (Figure 2A). We then expressed the mutants in HepG2 cells through lentivirus with a similar, low infection efficiency. Deletion of the JmjC domain or the CXXC Zinc finger domain abolished the suppression on PEPCK and G6Pase expression, whereas mutant lacking either the PHD domain or the F-box and Leucine-rich repeats remained fully functional (Figure 2A and Figure S3). Note that these Jhdm1a mutants were expressed at similar mRNA levels as their wild-type counterpart. The JmjC domain harbors the histone demethylation activity. Consistent with the effect of the JmjC deletion mutant, a demethylation-dead point mutant (H212A) [20] of Jhdm1a was no longer able to suppress PEPCK and G6Pase expression (Figure 2A and Figure S3). We next determined the effect of Jhdm1a on glucose production in vitro. We found that ectopic expression of wild type Jhdm1a, but not the demethylation defective mutants, inhibited glucose production in rat hepatoma FAO cells (Figure 2C). Taken together, these results demonstrate that both the demethylation activity and the CXXC Zinc finger domain of Jhdm1a are required for its negative modulation of gluconeogenic gene expression.

Bottom Line: In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level.Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity.This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus.

View Article: PubMed Central - PubMed

Affiliation: Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.

Show MeSH
Related in: MedlinePlus