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Molecular Endocrinology, doi:10.1210/me.2006-0307
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Molecular Endocrinology 21 (2): 550-563
Copyright © 2007 by The Endocrine Society

Insulin Represses Phosphoenolpyruvate Carboxykinase Gene Transcription by Causing the Rapid Disruption of an Active Transcription Complex: A Potential Epigenetic Effect

Robert K. Hall, Xiaohui L. Wang, Leena George, Stephen R. Koch and Daryl K. Granner

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Address all correspondence and requests for reprints to: Daryl K. Granner, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 707 Light Hall, Nashville, Tennessee 37232-0615. E-mail: daryl.granner{at}vanderbilt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin represses gluconeogenesis, in part, by inhibiting the transcription of genes that encode rate-determining enzymes, such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). Glucocorticoids stimulate expression of the PEPCK gene but the repressive action of insulin is dominant. Here, we show that treatment of H4IIE hepatoma cells with the synthetic glucocorticoid, dexamethasone (dex), induces the accumulation of glucocorticoid receptor, as well as many transcription factors, coregulators, and RNA polymerase II, on the PEPCK gene promoter. The addition of insulin to dex-treated cells causes the rapid dissociation of glucocorticoid receptor, polymerase II, and several key transcriptional regulators from the PEPCK gene promoter. These changes are temporally related to the reduced rate of PEPCK gene transcription. A similar disruption of the G-6-Pase gene transcription complex was observed. Additionally, insulin causes the rapid demethylation of arginine-17 on histone H3 of both genes. This rapid, insulin-induced, histone demethylation is temporally related to the disruption of the PEPCK and G-6-Pase gene transcription complex, and may be causally related to the mechanism by which insulin represses transcription of these genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE REGULATION OF gene expression is a fundamental mechanism by which insulin exerts its physiologic effects (1). Early reviews noted that as many as 150 genes are regulated by insulin directly, or indirectly, in a variety of tissues and cell lines (2). This number is surely an underestimation because a recent microarray analysis indicates that, during a hyperinsulinemic clamp, the expression of about 800 genes is affected in human skeletal muscle alone (3).

Insulin can repress and stimulate gene expression in the same cell, a phenomenon that is essential for its role in the coordination of complex metabolic processes. In one example of this type of simultaneous, multigenic control, insulin helps regulate glucose flux through the gluconeogenic and glycolytic pathways in the liver. The direction of the regulation depends on the metabolic status of the animal (4). In hyperglycemia, the expression of genes encoding the rate-determining enzymes of gluconeogenesis is negatively regulated by insulin, whereas the expression of the gene encoding glucokinase, a key glycolytic enzyme, is positively regulated by insulin (4, 5, 6). The opposite situation applies when the animal is hypoglycemic.

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-determining enzyme in the gluconeogenic pathway. This enzyme catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, which is the first committed step in gluconeogenesis (4). In contrast to many other metabolic enzymes, the activity of PEPCK is not regulated post-translationally or allosterically, but rather is directly proportional to the amount of PEPCK mRNA which, in turn, is primarily determined by the rate of transcription of the gene (7, 8, 9).

Transcription of the PEPCK gene is stimulated by glucocorticoids, glucagon (acting through cAMP), retinoic acid, and thyroid hormone in rat liver, isolated hepatocytes, and H4IIE cells (7, 10, 11). It is dominantly repressed by insulin in a protein synthesis-independent manner (12, 13). Most hormones, including those that stimulate PEPCK gene expression, initiate their action by promoting the binding of a transcription factor to a specific DNA sequence. Despite extensive searching, no consensus DNA element has been identified for either the positive or negative effects of insulin on gene transcription (1). Likewise, although several transcription factors have been implicated in the regulation of the PEPCK gene by insulin, including FoxO1 (14, 15, 16, 17, 18), FoxO3 (19), PGC-1{alpha} (20), CBP [cAMP response element (CRE) binding protein-(CREB)-binding protein] (21), and CCAAT/enhancer binding protein ß (C/EBPß) (22), none of these has been firmly established as the physiologically relevant mediator of the acute, protein synthesis-independent action of insulin on basal, or hormone-stimulated, PEPCK gene expression.

Given the difficulty in identifying one or more conserved insulin response sequences, or insulin regulated transcription factors that can account for all of the transcriptional effects of insulin, an approach in which the overall effect of insulin on a specific transcription complex is examined might yield clues as to the fundamental mechanism involved in insulin-regulated gene expression. This approach would allow for the detection of one or more effects of insulin on the transcription complex, including the addition or loss of DNA-protein or protein-protein interactions, the modification of proteins within the transcription complex, or changes in the modifications of histones associated with the gene.

Histones are known to play a dynamic role in eukaryotic gene transcription (23, 24, 25, 26). They can be modified by acetylation, phosphorylation, methylation, ubiquitination, sumoylation, and ADP ribosylation (27, 28). The most biologically relevant modifications appear to occur on the 20–35 residues in the N-terminal segment, or tail, of the histone (29), and a complex histone code has been proposed to explain the role of the various modifications in the regulation of gene expression (27). These modifications, or marks, may directly influence the interaction of DNA with the histones, which could then affect the ability of transcription factors to interact with their cognate DNA binding sites. In addition, certain histone marks attract proteins that may influence the transcription of a gene (29).

In this study, chromatin immunoprecipitation (ChIP) assays were used to measure changes in promoter occupancy of critical transcription factors on the PEPCK gene promoter when H4IIE cells are treated with dex, and then insulin, for various times. The presence or absence of certain modified histones was also monitored. The changes that occur to the PEPCK gene transcription complex under these hormonal conditions were compared with the rate of PEPCK gene transcription measured under the same treatment conditions. The data show that insulin causes the rapid disruption of the dex-induced PEPCK gene transcription complex. This process is accompanied by an acute decrease in the amount of acetylated histones associated with the PEPCK gene. Although histone deacetylation is typically involved in the repression of transcription, an inhibitor of histone deacetylase (HDAC) activity does not have any effect on the ability of insulin to repress PEPCK gene transcription. However, insulin causes a rapid decrease in the amount of PEPCK gene-associated histone H3 that is dimethylated at arginine-17 [H3-R17(diMe)]. The rate of disappearance of this mark tracks with the rate of insulin-induced repression of PEPCK gene transcription, thus the methylation status of H3-R17 may be important for PEPCK gene expression. The modification of this residue is typically associated with the coactivator-associated arginine methyltranferase 1 (CARM1) (30). However, in the case of the PEPCK gene, there is no correlation between the amounts of CARM1 and H3-R17(diMe) associated with the PEPCK gene. Similar findings were made in an analysis of the G-6-Pase gene. Given the rapidity of this demethylation on histone H3 (an observable decrease begins within 3–10 min of the initiation of insulin treatment), it is possible that insulin induces the activity of an H3-R17 demethylase, or inhibits the activity of an H3-R17 methyltransferase. The striking change in the integrity of the PEPCK and G-6-Pase gene transcription complex, and the concomitant changes in a specific histone mark, may be a critical step in the repression of gluconeogenic gene transcription by insulin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormonal Regulation of PEPCK Gene Transcription
H4IIE cells were transfected with a reporter plasmid (PEP/luc), which contains the luciferase gene driven by the PEPCK gene promoter sequence from –467 to +69 relative to the transcription initiation site. This region of the PEPCK gene promoter supports basal transcription, as well as transcription induced by glucocorticoids, cAMP, and retinoic acid (31, 32, 33, 34). The addition of insulin to H4IIE cells transfected with PEP/luc has no effect on the basal level of luciferase expression (Fig. 1AGo). The addition of dex causes a 5- to 6-fold increase of luciferase production and insulin represses the dex-induced expression of luciferase to, but not below, the basal level. This is in contrast to the regulation of the endogenous PEPCK gene where both basal and dex-induced PEPCK gene expression is virtually abolished by the addition of insulin to H4IIE cells [Fig. 1BGo (8)]. Thus, there appear to be regulatory mechanisms operative in the response of the endogenous PEPCK gene to insulin that are not effective on the transfected PEP/luc reporter gene.


Figure 1
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  		Fig. 1. Characterization of PEPCK Gene Expression in H4IIE Cells

A, H4IIE cells were transfected with PEP/luc and incubated for 18 h. The cells were then exposed to hormones for 4 h and luciferase activity was measured. B, H4IIE cells were exposed to hormones for 4 h, and PEPCK RNA was measured by real-time PCR. C–E, Cells were treated with hormones and nuclei were harvested at the indicated time intervals for the run-on transcription assay as described in Materials and Methods. C, Cells were treated with dex alone; D, insulin alone; E, cells were exposed to dex for 2 h and insulin was added for the indicated times before the end of the dex incubation period. The data illustrated represent the mean ± SEM of three or more independent experiments.

 
Transcription of the PEPCK gene was measured by the nuclear run-on assay, which provides a direct and sensitive determination of this process (35). With the addition of dex, transcription of the PEPCK gene rapidly increases until it reaches a maximum rate at 5 min (Fig. 1CGo). The rate of transcription remains relatively constant from 5–15 min, then declines slightly to reach a plateau about 60 min after the addition of dex. The addition of insulin to H4IIE cells causes a rapid reduction of the basal rate of transcription of the PEPCK gene, and by 30 min, transcription is virtually abolished [Fig. 1DGo (36)]. Insulin causes a similarly rapid repression of transcription in dex-treated H4IIE cells [Fig. 1EGo (7)]. These results indicate that the insulin-induced decrease of PEPCK mRNA observed in Fig. 1BGo is primarily due to a reduced rate of transcription.

Effect of Insulin on the Binding of Transcription Factors to the PEPCK Gene Promoter in Vivo
ChIP assays were performed to determine the effect of dex and insulin on the presence of various transcription factors on the PEPCK gene promoter in vivo. The results of a representative ChIP assay, and of a run-on transcription assay, are shown in Fig. 2AGo. There is a low, but reproducible, level of binding to the promoter by all of the monitored factors, including glucocorticoid receptor (GR), in the basal state. The presence of GR and the other factors on the PEPCK gene promoter under basal conditions is consistent with earlier observations which showed that the chromatin around the PEPCK promoter is in an open configuration, that the key DNA elements involved in basal and hormone-regulated transcription are all occupied to some extent, and that there is a detectable, basal rate of transcription of the gene (37, 38). Treatment of H4IIE cells with dex for 2 h increases the association of many of the transcription factors with the PEPCK gene promoter. The factors that exhibit the most dramatic dex-induced increase in promoter occupancy are GR, polymerase II (pol II), p300, CBP, Src-1, FoxO1, and FoxO3 (Fig. 2AGo). The amount of CREB, C/EBP{alpha}, C/EBPß, hepatic nuclear factor (HNF) 4{alpha}, and chicken ovalbumin upstream promoter transcription factor (COUP-TF) associated with the PEPCK gene promoter after dex treatment did not change significantly.


Figure 2
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  		Fig. 2. Disruption of the PEPCK Gene Promoter Transcription Complex by Insulin

H4IIE cells were treated with hormones as described in Fig. 1EGo. A, Representative ChIP assays are shown. A dot-blot from a typical transcription run-on assay is shown at the bottom of the figure for reference. B, Real-time PCR quantitation of the ChIP assay results. These data represent the mean values of at least three independent experiments. Error bars are omitted for clarity but did not exceed 10% of the mean.

 
Insulin was added to another set of cells 3, 10, and 30 min before the completion of a 2-h dex treatment, and ChIP assays were performed on cells isolated at these time points. The addition of insulin to dex-treated H4IIE cells causes a rapid dissociation of several proteins from the PEPCK gene promoter, although the extent and rapidity of dissociation of each factor differs (Fig. 2BGo). By 30 min, the amount of promoter bound GR, pol II, p300, CBP, FoxA2, FoxO1, and FoxO3 is at, or below, that measured in the absence of hormones (Fig. 2AGo). There is no significant dissociation of CREB, C/EBP{alpha}, C/EBPß, HNF4{alpha}, or COUP-TF from the promoter after insulin treatment. The presence of pol II and GR on the promoter correlates best with the rate of transcription (see bottom of Fig. 2AGo). This is especially apparent after 3 min of insulin treatment, when the association of pol II and GR with the promoter, and the transcription rate of the PEPCK gene, are relatively constant (Figs. 1EGo and 2AGo). By contrast, many of the other factors exhibit decreased association at the 3-min time point, particularly FoxO1 and FoxO3, which have already decreased to a level below that measured under basal conditions (Fig. 2AGo). This disconnection between transcription and promoter occupancy, particularly at the 3-min time point, is also notable for CBP, p300, Src-1, and FoxA2.

Effect of Insulin and a General Transcription Inhibitor on Promoter Occupancy by GR and Pol II
Because GR plays a central role in the assembly of the active PEPCK gene transcription complex, insulin could repress dex-induced transcription by reducing the amount of GR in the cell, perhaps by a proteolytic mechanism, or by inhibiting its ligand-induced import into the nucleus. However, neither the cellular concentration of GR, nor its translocation into the nucleus, are changed by the addition of insulin (Fig. 3AGo).


Figure 3
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  		Fig. 3. Effect of Insulin and DRB on GR Amount and Location

A, H4IIE cells were treated for 1 h with dex in the presence or absence of insulin. Cytoplasmic and nuclear extracts were prepared, and the amount and sub-cellular location of GR was determined by Western blot analysis. ns, A nonspecific band that appears on gels probed with GR antibody and thus serves as a loading control. B, Cells were treated with hormones and 10, 30, or 100 µM DRB for 2 h. RNA was isolated and PEPCK mRNA was quantified using real-time PCR. The data represent the mean ± SEM of at least three independent experiments. C, Cells were treated with hormones and 30 µM DRB for 2 h. A ChIP assay was then performed to determine the amount of pol II and GR associated with the PEPCK gene promoter. This is representative of three independent experiments.

 
The reduction of GR and pol II associated with the PEPCK gene promoter after insulin treatment might be the result of, rather than the cause of, transcription repression by insulin. To address this point H4IIE cells were treated with 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole (DRB), which is known to block transcription by inhibiting the phosphorylation of pol II by P-TEFb on Ser 2 of the pol II C-terminal domain (39). This phosphorylation event is required for efficient transcript initiation and elongation by pol II (40). As shown in Fig. 3BGo, DRB is a potent repressor of PEPCK gene expression. However, pol II and GR do not dissociate from the PEPCK gene promoter, even after a 2 h incubation of H4IIE cells with DRB (Fig. 3CGo). Therefore, the inhibition of transcription of the PEPCK gene per se is not sufficient to cause the dissociation of pol II and GR from the PEPCK gene promoter.

Promoter Occupancy by Transcription Factors on Other Genes Regulated by Insulin
Transcription of the G-6-Pase gene is, like the PEPCK gene, up-regulated by dex and repressed by insulin, and the expression of this gene correlates directly with the rate of gluconeogenesis (41) The promoter of the G-6-Pase gene has been less well characterized than that of the PEPCK gene, but it is known that GR and FoxO1 bind to this promoter upon dex treatment of H4IIE cells (42). We confirm this observation, and also show that the binding of CBP, p300, and pol II to the G-6-Pase gene promoter is enhanced by dex treatment (Fig. 4AGo). Insulin treatment causes a rapid dissociation of all these proteins from the G-6-Pase gene promoter, with disappearance rates similar to those noted for the PEPCK gene.


Figure 4
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  		Fig. 4. Effect of dex and dex Plus Insulin on the Transcription Complexes of the G-6-Pase Gene and Gene 33

H4IIE cells were treated as described in Fig. 1EGo, and ChIP assays were performed to detect factors bound to the G-6-Pase gene promoter (A) and the gene 33 promoter (B). These assays are representative of three or more independent experiments.

 
Transcription of gene 33 (also known as RALT and MIG6), an immediate early response gene, is stimulated by glucocorticoids and insulin in H4IIE cells (43, 44, 45). As shown in Fig. 4BGo, dex treatment causes an increase in the amount of pol II and GR associated with the gene 33 promoter and the addition of insulin does not significantly change this amount. These results are compatible with the stimulatory effect of insulin on gene 33 transcription, and clearly demonstrate that the binding behavior of these factors on this gene is different than that observed on the negatively regulated PEPCK and G-6-Pase gene promoters, measured over the same time frame in the same cell line.

Effect of Insulin on Acetylation of Histones Associated with the PEPCK Gene
Because the histone acetyl-transferases (HATs), CBP, p300, and Src-1, rapidly dissociate from the PEPCK gene promoter upon insulin treatment (see Fig. 2BGo), we reasoned that there might also be changes in the acetylation marks of substrate histones. CBP and p300 acetylate a number of lysine residues within the amino-terminal tails of histones H2A, H2B, H3, and H4 (46). The acetylase activity of these coregulators was determined by monitoring the degree of acetylation of lysine-14 on H3 (H3-K14) and acetylation of H4. Acetylation of lysine-9 on H3 (H3-K9) was also monitored because this residue is acetylated by Src-1, but it is a poor substrate for acetylation by CBP or p300 (47, 48). The acetylation of each of these marks is associated with active transcription (49). As shown in Fig. 5Go, A and B, the histones associated with the PEPCK gene are highly acetylated even under unstimulated conditions, which is consistent with the open chromatin configuration around the PEPCK gene promoter (38, 50). The amount of acetylation is not increased after dex treatment despite the recruitment of multiple HATs to the PEPCK gene promoter, so the histones associated with the PEPCK gene are constitutively acetylated, perhaps to the point of saturation. The degree of acetylation of these histone residues rapidly decreases after the addition of insulin to dex-treated H4IIE cells. The deacetylation of H3-K14 and H4 tracks with the dissociation of CBP and p300 from the PEPCK gene promoter, just as the deacetylation of H3-K9 parallels the loss of promoter-associated Src-1 (see Fig. 2BGo). Notably, the level of H3 associated with the PEPCK gene promoter remains constant throughout the time course of the experiment (Fig. 5Go).


Figure 5
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  		Fig. 5. Effect of Insulin on the Acetylation of Histones Associated with the PEPCK Gene

H4IIE cells were treated as described in Fig. 1EGo. A, This illustrates a representative ChIP assay in which the amount of total H3 and acetylated H3 and H4 associated with the PEPCK gene promoter is measured. B, The results were quantified by real-time PCR. The data represent the mean values of at least three independent experiments. Error bars are omitted for clarity but did not exceed 10% of the mean.

 
The possible role of deacetylation in insulin-induced repression of PEPCK gene transcription was addressed using sodium butyrate, a potent inhibitor of HDACs (51, 52). Treatment of H4IIE cells with sodium butyrate increases the rate of PEPCK gene transcription, but the relative changes of dex-induced activation and insulin-induced repression of transcription, are not affected (Fig. 6Go). As shown in Table 1Go, sodium butyrate prevents insulin-induced deacetylation of H3-K9, H3-K14, and H4. Thus, sodium butyrate blocks the activity of HDACs in H4IIE cells. Insulin does not affect this activity, but it still represses transcription of the PEPCK gene. These results indicate that histone deacetylation, per se, may not be responsible for acute insulin-induced repression of PEPCK gene transcription.


Figure 6
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  		Fig. 6. Effect of Sodium Butyrate on the Rate of Transcription of the PEPCK Gene

H4IIE cells were treated for 2 h with dex. During the last hour of this incubation the cells were treated with 3 mM sodium butyrate (hatched bars) or left untreated (solid bars). Insulin was added for the indicated times before the end of the 2 h dex treatment. Nuclei were isolated and run-on transcription assays were performed. The data are presented as the mean ± SEM of three or more independent experiments.

 

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  		Table 1. Ratio of Promoter-Associated, Acetylated Histones from Hormone- and Butyrate-Treated Cells, to that of Untreated Cells

 
Effect of Insulin on Methylation of Histones Associated with the PEPCK Gene
Histone methylation is also involved in the regulation of gene expression (53, 54). This modification was originally believed to be involved in long-term, epigenetic phenomena, but the recent discovery of lysine and arginine histone methyltransferases and demethylases, coupled with observations that histone methylation levels rapidly change on some genes in response to regulatory signals, raises the possibility that methylation, like acetylation and phosphorylation, may be involved in the acute regulation of gene expression (53). Thus, the methylation status of several residues on histones H3 and H4 was examined. A high ratio of H3 trimethylated-lysine 4 [H3-K4(triMe)] to dimethylated-lysine 4 [H3-K4(diMe)] at the promoter region of some genes has been linked to gene activation (55, 56). There is no change of either H3-K4(diMe) or H3-K4(triMe) associated with the PEPCK gene promoter in H4IIE cells treated with dex or dex plus insulin (Table 2Go). The methylation of H3-K9 is associated with transcription repression and gene silencing (57, 58). The amount of H3-K9(diMe) or H3-K9(triMe) also does not change with dex or dex plus insulin treatment (Table 2Go). Therefore, the methylation status of these lysine residues in H3 is apparently not involved in acute, insulin-induced repression of PEPCK gene transcription.


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  		Table 2. Ratio of Promoter-Associated, Methylated H3-K4 and H3-K9 from Hormone-Treated Cells to that of Untreated Cells

 
Protein methyltransferases 1 and 5 (PRMT1 and PRMT5) methylate arginine-3 of histone H4 (53). PRMT1 causes the asymmetric dimethylation of H4-R3 [H4-R3(aMe)], whereas PRMT5 causes the symmetric dimethylation of H4-R3 [H4-R3(sMe)] (53). The former modification is associated with transcription activation and the latter with repression (53). The amount of PRMT1 associated with the PEPCK gene promoter and its cognate histone modification, H4-R3(aMe) actually decrease when cells are treated with dex and decreases further after 30 min of dex plus insulin (Table 3Go). The association of PRMT5 with the PEPCK gene promoter paradoxically increases with dex treatment but does not change with insulin treatment (Table 3Go), and there is no significant change in H4-R3(sMe) during the time course of the experiment (Table 3Go). These results indicate that PRMT1 and PRMT5 are probably not involved in the regulation of PEPCK gene transcription by dex or insulin.


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  		Table 3. Ratio of Promoter-Associated Methyltransferases and their Cognate Methyl-Marks from Hormone-Treated Cells to that of Untreated Cells

 
CARM1, which methylates arginine-17 of histone H3 (H3-R17), is linked to transcription activation (53, 59). The amount of CARM1 on the PEPCK gene promoter is not significantly changed by either dex or dex plus insulin (Fig. 7Go). By contrast, the mark for this methyltransferase, H3-R17(diMe), changes significantly upon dex and dex plus insulin treatment (Fig. 7Go). The amount of this mark on histones associated with the PEPCK gene is extremely low in the absence of dex but increases 2.5-fold upon dex treatment. The amount of H3-R17(diMe) remains elevated after 3 min of insulin treatment in parallel with the rate of transcription, and pol II binding to the PEPCK promoter (see Figs. 1EGo and 2AGo). However, after 10 min, the amount of this modified histone on the promoter is back at the basal level (as is transcription, see Fig. 1EGo), and it remains low throughout the duration of insulin exposure. These findings indicate that insulin treatment results in the demethylation of H3-R17(diMe) and, because the amount of this histone mark associated with the PEPCK gene parallels the transcription rate of the PEPCK gene, it is possible that this histone modification may be important in the regulation of PEPCK gene transcription.


Figure 7
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  		Fig. 7. CARM1 and H3-R17(diMe) Associated with the PEPCK and G-6-Pase Genes

A typical ChIP assay for CARM1 and H3-R17 (diMe) is shown above the quantitative data, which is expressed as the mean ± SEM of three or more independent experiments. The data for the PEPCK gene is shown on the left and the G-6-Pase gene is shown on the right.

 
H3-R17 Methylation on the G-6-Pase Gene Promoter
The effect of insulin on the association of CARM1 with the G-6-Pase promoter and the demethylation of H3-R17(diMe) was determined because insulin also has a disruptive effect on the G-6-Pase gene transcription complex. The pattern of CARM1 associated with the G-6-Pase gene promoter is similar to that observed with the PEPCK gene promoter. The amount of H3-R17(diMe) associated with the promoter is rapidly reduced with the addition of insulin indicating that this histone mark is rapidly demethylated in the presence of insulin on two gluconeogenic enzyme genes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Despite much effort by many groups, including ours, the mechanism by which insulin represses PEPCK gene transcription remains elusive. A DNA element in the PEPCK gene promoter can confer insulin responsiveness to an unrelated reporter gene (60), and several experiments show that this element, referred to as the insulin response sequence (IRS), is involved in mediating at least part of the insulin response of the PEPCK gene (1). However, later experiments revealed that this element is not sufficient for the complete effect of insulin on PEPCK gene transcription. For example, insulin still causes significant repression of PEPCK gene expression when reporter gene constructs, in which this IRS is deleted, are randomly integrated into the genome of either stably transfected H4IIE cells or transgenic mice (1, 61, 62). The fact that insulin inhibits basal expression of the endogenous PEPCK gene, but is unable to exert this effect on reporter genes driven by the PEPCK gene promoter, provides another clue that the repression of PEPCK gene expression by insulin involves something more than a cis/trans mechanism involving a single element. Consideration of these results prompted us to examine the effect of insulin on the PEPCK gene transcription complex, including its effect on the local chromatin environment.

As mentioned in the introductory text, it has also not been possible to definitively link any single transcription factor to insulin-induced repression of PEPCK gene transcription. The FoxO proteins have attracted much attention in this regard because insulin is known to cause the egress of FoxO proteins from the nucleus (63, 64). Overexpression of FoxO3 in H4IIE cells stimulates expression of a reporter gene driven by a heterologous promoter in which the PEPCK gene IRS has been inserted, and this FoxO3-induced expression is repressed by insulin (19). In addition, FoxO1 or FoxO3 partially purified from bacteria as glutathione-S-transferase fusion proteins, bind the PEPCK IRS in vitro (14, 19). However, aH base-by-base mutation analysis of the IRS showed a dissociation of insulin repression of a transfected PEPCK gene promoter/reporter construct and FoxO3 binding in vitro (19). The conclusion from this study is that the FoxO proteins, at least in conjunction with the PEPCK IRS, are not the sole target of insulin action on this gene. This observation does not preclude the presence of another FoxO binding site that is more proximal to the transcription start site than the IRS, nor does it exclude a protein-protein interaction involving one or more of the FoxO proteins and other components of the PEPCK gene transcription complex.

The insulin-induced accumulation of a translational variant of C/EBPß, liver-enriched transcriptional inhibitory protein, has been linked to repression of the PEPCK gene by insulin (22). This factor competes with another translational variant of C/EBPß, liver-enriched activating protein, for binding to the CRE in the PEPCK gene promoter. ChIP assays revealed that, when liver-enriched transcriptional inhibitory protein is predominantly bound to the CRE, there is a reduction in the amount of CBP and pol II associated with the PEPCK gene promoter, thus connecting insulin with the binding of a transcriptional repressor to the PEPCK gene promoter (22). However, this mechanism of repression cannot account for the acute repression of transcription by insulin because it requires de novo protein synthesis.

In the present study, we show that insulin causes a very rapid disruption of the PEPCK gene transcription complex that is assembled in response to dex. A direct comparison of the composition of the PEPCK gene transcription complex with the rate of PEPCK gene transcription reveals some interesting points. Not surprisingly, the extent of pol II binding correlates best with transcription. The presence on the promoter of ligand-bound GR, which serves to nucleate the assembly of the fully active complex, also closely parallels the rate of transcription. Given its central role in the assembly of an active transcription complex, an effect of insulin on GR activity, amount, or cellular location could result in disassembly of the transcription complex. This clearly does not happen. Curiously, binding of the transcription coregulators p300, CBP, and Src-1, and an IRS-binding factor, FoxA2, is substantially reduced 3 min after the addition of insulin, when transcription is still at its maximal rate, and GR is still bound. Even more striking is the rapid dissociation of FoxO1 and FoxO3, which are essentially gone from the promoter after 3 min of insulin treatment. Although the disappearance of these factors from the PEPCK gene promoter precedes transcription repression, it is possible that the early dissociation of one or both of these proteins is the initial event that destabilizes the basic transcription complex leading to transcription repression. Identification of the initial insulin-induced event that triggers the destruction of the PEPCK gene transcription complex is of obvious importance.

There is specificity and selectivity of insulin-mediated disruption of transcription complexes. Most interesting in this regard is the fact that insulin causes a similarly rapid disruption of the G-6-Pase gene transcription complex. This gene, like the PEPCK gene, is induced by glucocorticoids and repressed by insulin, and both genes encode critical gluconeogenic enzymes. In contrast to the PEPCK gene promoter, the FoxO binding sites in the G-6-Pase gene promoter are much closer to the site of transcription initiation, and the collected evidence strongly favors an important role of the FoxO proteins in insulin action on the G-6-Pase gene (42). These differences have been, and remain, a topic worthy of further investigation. Insulin has the opposite effect on gene 33. Dex and insulin both increase gene 33 expression in H4IIE cells (43, 65). As shown here, dex treatment increases the binding of pol II and GR to the gene 33 promoter, and insulin does not reduce this amount. Thus, insulin has opposite effects on transcription, and on the integrity of transcription complexes of genes, in the same cell.

As noted above, insulin represses the basal expression of the endogenous PEPCK gene in H4IIE cells, but not that of a transfected PEPCK promoter/reporter gene in these cells. This observation, that transfected and endogenous genes are regulated differently, is not unique (66). The same phenomenon has been observed in an analysis of the promoters of the mouse mammary tumor virus (67), {alpha}1-antitrypsin (68), ß-globin (69), and c-jun (70, 71) genes. These observations have led investigators to conclude that the local chromatin environment of a gene plays an active role in regulating transcription. The arrangement of nucleosomes on and around a promoter, as well as modifications of histones within those nucleosomes, contributes to the regulation of transcription.

The acetylation of histones by HATs is usually associated with active gene transcription, whereas deacetylation of histones by HDACs is usually associated with repression of transcription (72, 73, 74). This was investigated as a potential mechanism for how insulin might repress PEPCK gene transcription, particularly because proteins with HAT activity, namely CBP, p300, and Src-1, are released from the PEPCK gene promoter when H4IIE cells are treated with insulin. Two lysine residues, K14 and K9 on the N-terminal tail of histone H3, were examined because of their strong association with active transcription. K14 is acetylated by p300, CBP, and Src-1, whereas K9 is preferentially acetylated by Src-1 (47, 48). The insulin-induced loss of these acetylation marks from the histone H3 associated with the PEPCK gene correlates with the insulin-induced dissociation of p300, CBP, and Src-1 from the PEPCK gene promoter. Sodium butyrate blocks the deacetylation of these marks by insulin, most likely due to the general inactivation of HDACs. Because sodium butyrate does not prevent the repression of basal or dex-induced PEPCK gene transcription by insulin, histone deacetylation does not appear to be involved in this mechanism.

In recent years, it has become apparent that the dynamic modification of histones by methylation/demethylation is associated with the regulation of gene transcription (53, 75). Mono-, di-, and trimethylation, in symmetric and asymmetric patterns, occurs on several lysine and arginine residues in histones H3 and H4 (53, 75). As described above, several of the enzymes responsible for these modifications have been identified, and the modifications themselves have been associated with activated or repressed transcription. We analyzed many of these methylation targets and found no correlation of H3-K4(diMe or triMe) or H3-K9(diMe or triMe) with PEPCK gene regulation by either dex or insulin. We also saw no correlation of asymmetric methylation of H4-R3 by protein arginine methyltransferase 1 (PRMT1), or the symmetric methylation of H4-R3 by PRMT5, as a result of treatment with these hormones.

One methylation/demethylation event does correlate with PEPCK gene transcription. The amount of H3-R17(diMe) associated with the PEPCK gene increases upon dex treatment of H4IIE cells and decreases with the subsequent addition of insulin, in a manner that parallels the rate of PEPCK gene transcription. This demethylation event is also observed on the G-6-Pase gene promoter. The methyltransferase CARM1 catalyzes the methylation of H3-R17 (59) and was initially identified because of its ability to interact with the p160 family of nuclear receptor coactivator proteins, and to act as a coactivator with the estrogen receptor (30). CARM1 also interacts with a broad range of transcription factors including CBP/p300 (76), ß-catenin (77), MEF2C (78), p53 (79), nuclear factor-{kappa}B (80), and CREB (81). Despite the strong correlation between CARM1 and its ability to dimethylate H3-R17, the amount of CARM1 associated with the PEPCK and G-6-Pase gene promoters does not change in response to dex or dex plus insulin. This observation could be explained in at least three ways: 1) CARM1 could be activated by dex, and inactivated by insulin, without leaving the promoter; 2) the extent of H3-R17 methylation could be regulated by the recruitment of a demethylase, or by an altered activity of an already bound demethylase; or 3) the methylation status of H3-R17, in the context of these gene promoters, may be controlled by a methyltransferase other than CARM1, as has been observed in the context of the GADD45 gene (79). In this case, recruitment of CARM1 to the GADD45 gene promoter also does not temporally correlate with the appearance of dimethylated H3-R17.

A family of enzymes called peptidylarginine deiminases (PADs) convert arginine and mono-methyl arginine to citrulline. One of these enzymes, PAD4, may play a role in the demethylation of H3-R17 (82, 83, 84, 85, 86, 87). However, because CARM1 causes dimethylation of H3-R17, two or more PADs, or a PAD acting in concert with another enzyme, are probably responsible for complete demethylation of this residue (84, 87). Studies of the regulation of the pS2 gene by estrogen are intriguing in this regard (83, 84, 85, 88, 89). CARM1 and/or PRMT1 are recruited to the pS2 gene promoter upon binding of the liganded estrogen receptor to its cognate DNA binding site. The increase of H3-R17 methylation and the activation of pS2 gene transcription occur concomitantly. Removal of the ligand (estradiol) results in the dissociation of estrogen receptor from the promoter, the association of PAD4 with the promoter, and a reduction of H3-R17 methylation. It has been postulated that the conversion of methylated arginine to citrulline induces a conformational change of a critical nucleosome that results in the repression of pS2 gene transcription (84). We could not address the question of whether or not insulin induces the recruitment or activation of PAD4 due to the lack of adequate antibodies to rat PAD4.

Insulin counteracts the activation of transcription of the PEPCK gene by multiple hormones, and it represses basal transcription as well. It could accomplish this through several separate mechanisms, but a more parsimonious explanation is that it disrupts a common mechanism of gene transcription, shared by all activating signals. The data presented here clearly show that insulin causes the rapid dissociation of several components of the PEPCK gene transcription complex from the promoter. Although this could be initiated by a classical cis/trans mechanism, such is not a necessary requirement. One can imagine an epigenetic action, perhaps a critical histone methylation/ demethylation event, that destabilizes the entire complex, the components of which then leave the promoter in a hierarchical sequence. The definition of the primary target of the insulin signal transduction pathway on the PEPCK gene promoter could provide a novel approach to controlling gluconeogenesis, which is not normally restrained by insulin in type 2 diabetes mellitus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture, Hormone Treatments, Transfection, and mRNA Measurement
H4IIE rat hepatoma cells were propagated in low glucose-DMEM supplemented with glutamine and 10% (vol/vol) serum (2% newborn, 3% calf, and 5% fetal bovine) and kept at 37 C in a 5% CO2 atmosphere. The cells were incubated in low glucose-DMEM with no serum (DMEM-0) overnight before hormone treatment. Unless otherwise stated, hormone treatments were performed as follows: on the day of the experiment, the cells were exposed to 500 nM dexamethasone (Sigma) for a total of 2 h. Insulin (Sigma) was added to the dex-containing medium at a final concentration of 10 nM at 90, 110, and 117 min after initiation of the dex treatment providing the 30-, 10-, and 3-min insulin time points, respectively. The plasmid PEP/luc contains the luciferase reporter gene driven by the PEPCK gene promoter sequence from –467 to +65 and its construction was described previously (22). H4IIE cells were transfected as described previously (32), and the measurement of PEPCK mRNA from the endogenous gene was performed essentially as described previously (90).

Nuclear Run-On Transcription Assay
The nuclear run-on assay was performed essentially as described (7, 8) with the following exceptions: 1) The labeled RNA was isolated with a QIAGEN RNeasy kit according to the manufacturer’s instructions; 2) Zeta probe filters (Bio-Rad, Hercules, CA) were used in place of nitrocellulose and; 3) The plasmid p.R3 was made by cloning the 5.8-kb EcoRI fragment of the PEPCK gene from p{lambda}PC112.R3 (7), into the EcoRI site of pSP73 (Promega). This plasmid was used for hybridization of labeled RNA obtained from the run-on experiment. The empty vector, pSP73 was used to measure background hybridization.

ChIP Assays
ChIP assays were performed essentially as described previously (90) with some modifications. Formaldehyde cross-linking was carried out for 10 min at room temperature to probe occupancy of the PEPCK promoter by transcription factors. The cross-linking time was reduced to 3 min for the analysis of histone binding. The protein-A or protein-G agarose beads pretreated with sonicated salmon sperm DNA were obtained from Upstate (Lake Placid, NY). The primers specific for the PEPCK gene promoter were 5'-GTTTCACGTCTCAGAGCTGA and 5'-ACCGTGACTGTTGCTGATGC. Primers for the G-6-Pase gene promoter were 5'-CAGACTCTGCCCTGAGCCTCTGGCCTG and 5'CCCTGGATTCAGTCTGTA GGTCAACCTAGC. Primers for the Gene 33 promoter were 5'-CTCAGGAGACGAGG ATGC and 5'-TATAAGGCGAGCCGACAAC. The antibodies used for ChIP assays were as follows: GR (M-20), pol II (C-21), p300 (N-15), CBP (A-22), HNF4{alpha} (C-19), COUP-TF (N-19), C/EBP{alpha} (14AA), C/EBPß (C-19), and Src-1 (M-341) from Santa Cruz; FoxA2, di- and trimethyl H3-K4, di- and trimethyl H3-K9, PRMT1, PRMT5, and symmetric dimethyl H4-R3 from Abcam; FoxO1 was from Cell Signaling; FoxO3, pan H3, acetylated H3-K9, acetylated H3-K14, acetylated H4, CARM1, dimethyl H4-R3, and dimethyl H3-R17 were obtained from Upstate.


   ACKNOWLEDGMENTS
 
The authors thank Michael Stallcup and Tony Weil for critical reading of the manuscript, and Deborah C. Brown for her help in preparing this manuscript.


   FOOTNOTES
 
Current address for X.L.W.: Joslin Diabetes Center and Harvard University Medical School, One Joslin Place, Boston, Massachusetts 02215.

This work was supported by grants from the Joe C. Davis Foundation, the Veterans Affairs Research Service, and National Institutes of Health Grants DK35107 and DK20593 (Vanderbilt Diabetes Research and Training Center) (to D.K.G.).

Disclosure Summary: R.K.H., X.L.W., L.G., and S.R.K. have nothing to disclose. D.K.G. is on the Board of, and has an equity interest in, OSI Pharmaceuticals and is a consultant to its subsidiary, Prosidion, Ltd.

First Published Online November 9, 2006

Abbreviations: CARM1, Coactivator-associated arginine methyltransferase-1; CBP, CREB binding protein; C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; COUP-TF, chicken ovalbumin upstream promoter transcription factor; CRE, cAMP response element; CREB, CRE binding protein; dex, dexamethasone; DRB, 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole; G-6-Pase, glucose-6-phosphatase; GR, glucocorticoid receptor; HATs, histone acetyl-transferases; HDAC, histone deacetylase; HNF, hepatic nuclear factor; IRS, insulin response sequence; PAD, peptidylarginine deiminase; PEPCK, phosphoenolpyruvate carboxykinase; pol II, polymerase II; PRMT1 and PRMT5, protein methyltransferases 1 and 5.

Received for publication July 28, 2006. Accepted for publication October 30, 2006.


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