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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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Related in: MedlinePlus

Jhdm1a regulates the expression of C/EBPα, thereby indirectly modulating gluconeogenic gene expression.(A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.
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pgen-1002761-g004: Jhdm1a regulates the expression of C/EBPα, thereby indirectly modulating gluconeogenic gene expression.(A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.

Mentions: We explored how Jhdm1a regulates gluconeogenesis. We initially speculated that Jhdm1a might associate with the transcriptional regulator complex on the promoters of PEPCK and G6Pase and directly regulate their expression. To test this idea, we performed chromatin immunoprecipitation experiments in HepG2 cells ectopically expressing HA-tagged Jhdm1a. Unexpectedly, Jhdm1a did not associate with either PEPCK promoter or G6Pase promoter (Figure S9). The promoter regions we examined have been well characterized previously and are subjected to extensive regulation by an array of transcription regulators [6]–[9]. The lack of association of Jhdm1a with PEPCK and G6Pase promoters indicates to us that Jhdm1a might not directly regulate the expression of these two genes. We thus considered a possibility that Jhdm1a instead regulates the expression of any of the involved transcription factors or co-factors [10], [11]. We knocked down Jhdm1a in HepG2 cells and examined their expression. We found that the transcription factor C/EBPα was the only one whose expression level was significantly increased (Figure 4A). Similarly, knockdown of Jhdm1a promoted C/EBPα expression in primary mouse hepatocytes (Figure 4B). As a result of increased C/EBPα level, the association of C/EBPα with its binding sites within the PEPCK and G6Pase promoters was strongly enhanced in Jhdm1a knockdown HepG2 cells (Figure 4C). Members of C/EBPs were shown to activate the expression of PEPCK and G6Pase in vitro [24], [25]. We confirmed these previous results and also observed a remarkably similar target gene expression pattern between Jhdm1a silencing and C/EBPα ectopic expression (comparing Figure 1C and Figure 4D), supporting a functional connection between Jhdm1a and C/EBPα.


The histone demethylase Jhdm1a regulates hepatic gluconeogenesis.

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

Jhdm1a regulates the expression of C/EBPα, thereby indirectly modulating gluconeogenic gene expression.(A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375226&req=5

pgen-1002761-g004: Jhdm1a regulates the expression of C/EBPα, thereby indirectly modulating gluconeogenic gene expression.(A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.
Mentions: We explored how Jhdm1a regulates gluconeogenesis. We initially speculated that Jhdm1a might associate with the transcriptional regulator complex on the promoters of PEPCK and G6Pase and directly regulate their expression. To test this idea, we performed chromatin immunoprecipitation experiments in HepG2 cells ectopically expressing HA-tagged Jhdm1a. Unexpectedly, Jhdm1a did not associate with either PEPCK promoter or G6Pase promoter (Figure S9). The promoter regions we examined have been well characterized previously and are subjected to extensive regulation by an array of transcription regulators [6]–[9]. The lack of association of Jhdm1a with PEPCK and G6Pase promoters indicates to us that Jhdm1a might not directly regulate the expression of these two genes. We thus considered a possibility that Jhdm1a instead regulates the expression of any of the involved transcription factors or co-factors [10], [11]. We knocked down Jhdm1a in HepG2 cells and examined their expression. We found that the transcription factor C/EBPα was the only one whose expression level was significantly increased (Figure 4A). Similarly, knockdown of Jhdm1a promoted C/EBPα expression in primary mouse hepatocytes (Figure 4B). As a result of increased C/EBPα level, the association of C/EBPα with its binding sites within the PEPCK and G6Pase promoters was strongly enhanced in Jhdm1a knockdown HepG2 cells (Figure 4C). Members of C/EBPs were shown to activate the expression of PEPCK and G6Pase in vitro [24], [25]. We confirmed these previous results and also observed a remarkably similar target gene expression pattern between Jhdm1a silencing and C/EBPα ectopic expression (comparing Figure 1C and Figure 4D), supporting a functional connection between Jhdm1a and C/EBPα.

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