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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 C/EBPα expression by Jhdm1a is mediated by USF1.(A) Jhdm1a associates with putative USF1-binding sites on the C/EBPα promoter region. Adenoviral HA-Jhdm1a were expressed in HepG2 cells and ChIP assays were performed with HA antibody. Data were shown from one representative of three experiments with similar results. (B) Jhdm1a interacts with USF1. Hela cells were co-transfected with indicated plasmids. Cell extracts were incubated with HA beads and immunoprecipitates were probed with Flag antibody. (C) HA-Jhdm1a (adenoviral) along with shRNA (lentiviral) against USF1 was co-expressed in HepG2 cells. ChIP assays were performed with HA antibody. (D) Gene expression in HepG2 cells expressing lentiviral USF1 shRNA. Data were shown from one representative of three experiments.
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pgen-1002761-g005: Suppression of C/EBPα expression by Jhdm1a is mediated by USF1.(A) Jhdm1a associates with putative USF1-binding sites on the C/EBPα promoter region. Adenoviral HA-Jhdm1a were expressed in HepG2 cells and ChIP assays were performed with HA antibody. Data were shown from one representative of three experiments with similar results. (B) Jhdm1a interacts with USF1. Hela cells were co-transfected with indicated plasmids. Cell extracts were incubated with HA beads and immunoprecipitates were probed with Flag antibody. (C) HA-Jhdm1a (adenoviral) along with shRNA (lentiviral) against USF1 was co-expressed in HepG2 cells. ChIP assays were performed with HA antibody. (D) Gene expression in HepG2 cells expressing lentiviral USF1 shRNA. Data were shown from one representative of three experiments.

Mentions: To identify the molecular mechanism by which Jhdm1a regulates C/EBPα expression, we first examined whether Jhdm1a associates with the C/EBPα locus. The C/EBPα locus contains a single exon. We expressed HA-tagged Jhdm1a in hepatic cells and performed chromatin immunoprecipitation experiments using antibody against the HA tag. We found that Jhdm1a was associated with the C/EBPα promoter region but not with the intragenic region (Figure 5A). Interestingly, this promoter region contains four separate transcription factor USF1 binding sites that have been implicated in C/EBPα expression [30], [31] and Jhdm1a was present on three of them. An interaction between Jhdm1a and USF1 was readily detected in cells expressing both of them (Figure 5B). Moreover, reduction of USF1 level by shRNA-mediated silencing diminished the association of exogenous Jhdm1a with these sites (Figure 5C). Despite the high background of the Jhdm1a antibody, we were also able to show that endogenous Jhdm1a associated with the USF1 binding sites, since knockdown of Jhdm1a decreased its association with these sites (Figure S11). Functionally, knockdown of USF1 led to an increase of C/EBPα expression and accordingly, an increase of PEPCK expression (Figure 5D). These data suggest a model in which USF1 recruits Jhdm1a to the C/EBPα promoter to negatively regulate its 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 C/EBPα expression by Jhdm1a is mediated by USF1.(A) Jhdm1a associates with putative USF1-binding sites on the C/EBPα promoter region. Adenoviral HA-Jhdm1a were expressed in HepG2 cells and ChIP assays were performed with HA antibody. Data were shown from one representative of three experiments with similar results. (B) Jhdm1a interacts with USF1. Hela cells were co-transfected with indicated plasmids. Cell extracts were incubated with HA beads and immunoprecipitates were probed with Flag antibody. (C) HA-Jhdm1a (adenoviral) along with shRNA (lentiviral) against USF1 was co-expressed in HepG2 cells. ChIP assays were performed with HA antibody. (D) Gene expression in HepG2 cells expressing lentiviral USF1 shRNA. Data were shown from one representative of three experiments.
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Related In: Results  -  Collection

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

pgen-1002761-g005: Suppression of C/EBPα expression by Jhdm1a is mediated by USF1.(A) Jhdm1a associates with putative USF1-binding sites on the C/EBPα promoter region. Adenoviral HA-Jhdm1a were expressed in HepG2 cells and ChIP assays were performed with HA antibody. Data were shown from one representative of three experiments with similar results. (B) Jhdm1a interacts with USF1. Hela cells were co-transfected with indicated plasmids. Cell extracts were incubated with HA beads and immunoprecipitates were probed with Flag antibody. (C) HA-Jhdm1a (adenoviral) along with shRNA (lentiviral) against USF1 was co-expressed in HepG2 cells. ChIP assays were performed with HA antibody. (D) Gene expression in HepG2 cells expressing lentiviral USF1 shRNA. Data were shown from one representative of three experiments.
Mentions: To identify the molecular mechanism by which Jhdm1a regulates C/EBPα expression, we first examined whether Jhdm1a associates with the C/EBPα locus. The C/EBPα locus contains a single exon. We expressed HA-tagged Jhdm1a in hepatic cells and performed chromatin immunoprecipitation experiments using antibody against the HA tag. We found that Jhdm1a was associated with the C/EBPα promoter region but not with the intragenic region (Figure 5A). Interestingly, this promoter region contains four separate transcription factor USF1 binding sites that have been implicated in C/EBPα expression [30], [31] and Jhdm1a was present on three of them. An interaction between Jhdm1a and USF1 was readily detected in cells expressing both of them (Figure 5B). Moreover, reduction of USF1 level by shRNA-mediated silencing diminished the association of exogenous Jhdm1a with these sites (Figure 5C). Despite the high background of the Jhdm1a antibody, we were also able to show that endogenous Jhdm1a associated with the USF1 binding sites, since knockdown of Jhdm1a decreased its association with these sites (Figure S11). Functionally, knockdown of USF1 led to an increase of C/EBPα expression and accordingly, an increase of PEPCK expression (Figure 5D). These data suggest a model in which USF1 recruits Jhdm1a to the C/EBPα promoter to negatively regulate its 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