Aspects of the Control of Phosphoenolpyruvate Carboxykinase Gene Transcription

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-C) 2The abbreviations used are: PEPCK-Ccytosolic phosphoenolpyruvate carboxykinaseBt2cAMPdibutyryl cAMPGRUglucocorticoid regulatory unitPPARγ2peroxisome proliferator-activated receptor-γ2LCRlocus control region. occupies an important position in energy homeostasis because it is involved in the regulation of glucose synthesis, fatty acid re-esterification, and the cataplerosis of citric acid cycle anions. PEPCK-C gene transcription can change very rapidly; cAMP administration causes a 10-fold increase in transcription in liver within 20 min (1Lamers W.H. Hanson R.W. Meisner H.M. Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 5137-5141Crossref PubMed Scopus (307) Google Scholar), whereas insulin rapidly inhibits transcription (i.e. a 50% decrease in PEPCK-C mRNA levels within 30 min) (2Granner D. Andreone T. Sasaki K. Beale E. Nature. 1983; 305: 549-551Crossref PubMed Scopus (265) Google Scholar). In contrast, the activity of the enzyme changes far less rapidly, i.e. a 2-fold increase in hepatic PEPCK-C activity was noted 3 h after Bt2cAMP administration (3Reshef L. Hanson R.W. Biochem. J. 1972; 127: 809-818Crossref PubMed Scopus (25) Google Scholar). This suggests regulation of PEPCK-C mRNA processing or post-translational modification of the enzyme. A recent study using both yeast and HepG2 hepatoma cells has demonstrated that PEPCK-C is post-translationally regulated by acetylation at Lys-19 and Lys-514 of PEPCK-C; this increases PEPCK-C activity in yeast and hepatoma cells (4Lin Y.Y. Lu J.Y. Zhang J. Walter W. Dang W. Wan J. Tao S.C. Qian J. Zhao Y. Boeke J.D. Berger S.L. Zhu H. Cell. 2009; 136: 1073-1084Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). However, it is generally true that the induced changes in the amount of immunoreactive PEPCK-C protein correlate well with the corresponding alterations in the activity of the enzyme (5Hopgood M.F. Ballard F.J. Reshef L. Hanson R.W. Biochem. J. 1973; 134: 445-453Crossref PubMed Scopus (78) Google Scholar). cytosolic phosphoenolpyruvate carboxykinase dibutyryl cAMP glucocorticoid regulatory unit peroxisome proliferator-activated receptor-γ2 locus control region. PEPCK-C is a single copy gene whose transcription is regulated by the binding of transcription factors to specific sites on the gene promoter. Fig. 1 summarizes the location of transcription factor-binding sites that, together with the transcription factors and co-regulators, influence the expression of the gene in mammalian tissues. Regulatory elements in the PEPCK-C gene promoter may best be understood as “units,” each of which is composed of several different transcription factor-binding sites that function together in response to hormonal and dietary stimuli (Fig. 1B). These include the cAMP regulatory unit in liver (6Roesler W.J. Mol. Cell. Endocrinol. 2000; 162: 1-7Crossref PubMed Scopus (71) Google Scholar), the acidosis regulatory unit in kidney (7Taylor L. Curthoys N.P. Biochem. Mol. Biol. Educ. 2004; 32: 291-304Crossref PubMed Scopus (101) Google Scholar), and the GRU, which is critical for the response of the gene to glucocorticoids and insulin in liver (8Imai E. Stromstedt P.E. Quinn P.G. Carlstedt-Duke J. Gustafsson J.A. Granner D.K. Mol. Cell. Biol. 1990; 10: 4712-4719Crossref PubMed Scopus (253) Google Scholar, 9Cassuto H. Kochan K. Chakravarty K. Cohen H. Blum B. Olswang Y. Hakimi P. Xu C. Massillon D. Hanson R.W. Reshef L. J. Biol. Chem. 2005; 280: 33873-33884Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). PEPCK-C is robustly expressed in four tissues (liver, kidney, and white and brown adipose), and several hormones differentially regulate its transcription in a tissue-specific manner. The rate of transcription of the gene for PEPCK-C is altered rapidly and differentially in these tissues in response to changes in the dietary and hormonal status of the animal. Thus, a stimulus that induces gene transcription in one tissue might repress it in another. One important caveat is that low levels of PEPCK-C are present in many mammalian tissues (10Zimmer D.B. Magnuson M.A. J. Histochem. Cytochem. 1990; 38: 171-178Crossref PubMed Scopus (59) Google Scholar), but there is very little information concerning the factors that control transcription of the gene in tissues other than the four listed above. Finally, PEPCK-C in mammals first appears at birth (11Ballard F.J. Hanson R.W. Biochem. J. 1967; 104: 866-871Crossref PubMed Scopus (309) Google Scholar), an event that contributes to the initiation of gluconeogenesis and glyceroneogenesis in the newborn. The regulation of transcription of the PEPCK-C gene had been reviewed in detail previously (12Hanson R.W. Reshef L. Annu. Rev. Biochem. 1997; 66: 581-611Crossref PubMed Scopus (641) Google Scholar, 13Chakravarty K. Cassuto H. Reshef L. Hanson R.W. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 129-154Crossref PubMed Scopus (184) Google Scholar). This minireview focuses on areas that have received less attention over the years. The mechanisms involved in the tissue-specific expression of PEPCK-C and the pattern of its expression during development are critical areas for future investigation. PEPCK-C activity is present at significant levels in the periportal region of the liver, kidney cortex, and brown and white adipose tissue, all known to be involved in either gluconeogenesis or glyceroneogenesis. However, an immunohistochemical analysis of PEPCK-C in mouse by Zimmer and Magnuson (10Zimmer D.B. Magnuson M.A. J. Histochem. Cytochem. 1990; 38: 171-178Crossref PubMed Scopus (59) Google Scholar) detected the enzyme in a wide variety of mouse tissues, most of which do not make glucose and store little triglyceride (i.e. bladder, stomach, small intestine, vagina, parotid gland, submaxillary gland, and brain). During mouse development, immunoreactive PEPCK-C was observed in the nervous system and in somites of 15-day mouse embryos (10Zimmer D.B. Magnuson M.A. J. Histochem. Cytochem. 1990; 38: 171-178Crossref PubMed Scopus (59) Google Scholar). PEPCK-C is largely absent in mammalian liver before birth and develops to greater than adult levels in rats livers within 8 h after birth (14Philippidis H. Hanson R.W. Reshef L. Hopgood M.F. Ballard F.J. Biochem. J. 1972; 126: 1127-1134Crossref PubMed Scopus (46) Google Scholar); this parallels the initiation of hepatic gluconeogenesis. The factors that control the tissue-specific development of PEPCK-C gene expression are poorly understood (Fig. 1C). It is likely that the developmentally regulated expression of key transcription factors in specific tissues patterns the transcription of the gene for PEPCK-C. As an example, deleting the gene for CAAT/enhancer-binding protein-α in mouse completely represses expression of PEPCK-C in liver at birth and leads to profound hypoglycemia in the newborn (15Wang N.D. Finegold M.J. Bradley A. Ou C.N. Abdelsayed S.V. Wilde M.D. Taylor L.R. Wilson D.R. Darlington G.J. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (850) Google Scholar). This shifts the emphasis from the PEPCK-C gene itself to factors that control expression of genes for transcription factors during development. The transcriptional control of PEPCK-C gene expression becomes prominent during development (especially during the perinatal period) when the gene undergoes dramatic changes from dormancy to being actively transcribed. This involves chromatin remodeling, which has been studied by determining (a) the methylation status of the DNA, (b) the chromatin status, and (c) the methylation of Arg-17 in histone H3. Benvenisty et al. (16Benvenisty N. Mencher D. Meyuhas O. Razin A. Reshef L. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 267-271Crossref PubMed Scopus (75) Google Scholar) analyzed the extent of methylation of 18 sites in the PEPCK-C gene and its surrounding sequences in adult liver, kidney, spleen, and heart and in fetal liver using CpG site-sensitive enzymes. There was extensive undermethylation of the gene in adult liver and kidney (both actively express PEPCK-C), whereas the gene in spleen, heart, and fetal liver (tissues that do not express PEPCK-C) was heavily methylated, suggesting that the level of methylation is directly related to the expression of the gene. Injection of 5-azacytidine (which decreases methylase activity) into 19-day rat fetuses caused demethylation of the gene (after 48 h) and a marked increase in PEPCK-C mRNA in liver but not in spleen (16Benvenisty N. Mencher D. Meyuhas O. Razin A. Reshef L. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 267-271Crossref PubMed Scopus (75) Google Scholar). Bt2cAMP induced the premature transcription of the PEPCK-C gene in liver within 3 h, whereas demethylation of the gene was observed 2 days later (17Benvenisty N. Reshef L. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1132-1136Crossref PubMed Scopus (32) Google Scholar). Although these studies suggest that changes in the pattern of methylation of the PEPCK-C gene locus accompany the developmental and tissue-specific expression of PEPCK-C, other activating factors, which are likely not sensitive to methylation status, are clearly involved in this process. The PEPCK-C gene retains a compact structure, extremely DNase I-resistant, in fetal rat liver until the perinatal period. However, intra-uterine injection of Bt2cAMP into 19-day rat fetuses for 3 h induced not only PEPCK-C gene transcription but concomitantly chromatin associated with the gene-acquired sensitivity to DNase I (17Benvenisty N. Reshef L. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1132-1136Crossref PubMed Scopus (32) Google Scholar). The partial DNase I sensitivity of the PEPCK-C chromatin elicited by Bt2cAMP is in accord with the hepatocytes composing only 35% of the entire fetal liver cell population at this gestational age (18Greengard O. Federman M. Knox W.E. J. Cell Biol. 1972; 52: 261-272Crossref PubMed Scopus (226) Google Scholar). Changesin the methylation status of histone H3/H4 have been shown to modulate the transcription and chromatin occupation of transcription factors in the PEPCK-C and glucose-6-phosphatase genes. Hall et al. (19Hall R.K. Wang X.L. George L. Koch S.R. Granner D.K. Mol. Endocrinol. 2007; 21: 550-563Crossref PubMed Scopus (66) Google Scholar) noted that ablation of the insulin-responsive sequence failed to prevent the dominant-negative response to insulin in liver, as would be predicated if this area of the gene promoter were the only factor involved in the negative effect of insulin on transcription. They found a gradual but rather rapid disappearance of many key transcription factors from the chromatin of the genes for both PEPCK-C and glucose-6-phosphatase within 3 min after the addition of insulin to hepatoma cells; this correlated with a parallel decrease in methylated Arg-17 residues from histone H3 and with the rate of transcription of the two genes. It is thus possible that demethylation of the histone H3 arginine residue underlies the repression of gene transcription by insulin. This is an attractive hypothesis because it might explain the rapid and dominant-negative effect of insulin on PEPCK-C gene transcription. It thus would be interesting to assess, using the livers of 19-day rat fetuses, when the chromatin for PEPCK-C is in a compact conformation, whether histone H3 Arg-17 is demethylated, and whether it becomes methylated upon birth, when the gene is initially transcribed. In addition to the developmental acquisition of global DNase I-sensitive chromatin, Ip et al. (20Ip Y.T. Granner D.K. Chalkley R. Mol. Cell. Biol. 1989; 9: 1289-1297Crossref PubMed Scopus (36) Google Scholar) identified five distinct DNase I-hypersensitive sites in the PEPCK-C gene in rat liver-derived hepatoma cells (Fig. 2A). Site A mapped to a 220-bp region centered at −4800 bp; Site B mapped at about −1300 bp; Site C mapped between −370 to −30 bp; Site D mapped at +4650 bp (within intron 8 of the gene); and Site E is composed of two closely spaced sites at +6100 and +6300 bp, which is within the 3′-untranslated region of the PEPCK-C gene (all sites are numbered relative to the transcription start site, i.e. +1). Several of the DNase I-hypersensitive sites have been linked to the tissue-specific transcription of the PEPCK-C gene. Williams et al. (21Williams C.P. Postic C. Robin D. Robin P. Parrinello J. Shelton K. Printz R.L. Magnuson M.A. Granner D.K. Forest C. Chalkley R. Mol. Cell. Endocrinol. 1999; 148: 67-77Crossref PubMed Scopus (11) Google Scholar) noted that hypersensitive Site A (at −4800 bp) is strictly liver-specific and is absent in kidney and NIH3T3–442A preadipocytes and adipocytes. Another hypersensitive site at about −1100 bp of the PEPCK-C gene promoter (termed Site B) is present in NIH3T3–442A cells (which differentiate into adipocytes) but not in NIH3T3-C2 cells (which do not differentiate into adipocytes); this site was not detected in mouse liver. This region of the PEPCK-C gene promoter contains a PPARγ2-binding site that is critical for the adipose tissue-specific expression of the gene (Figs. 1C and 2B) (22Devine J.H. Eubank D.W. Clouthier D.E. Tontonoz P. Spiegelman B.M. Hammer R.E. Beale E.G. J. Biol. Chem. 1999; 274: 13604-13612Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is thus likely that an alteration in the chromatin conformation in this region of the PEPCK-C gene promoter is involved in the control of adipose tissue-specific expression of the gene. Cissell and Chalkley (23Cissell M.A. Chalkley R. Biochim. Biophys. Acta. 1999; 1445: 299-313Crossref PubMed Scopus (5) Google Scholar) examined the tissue-specific chromatin structure of the PEPCK-C gene in kidney and liver. They detected several DNase I-hypersensitive sites in the gene in kidney. As expected, one of the sites mapped to between −370 and −30 bp relative to the start site of transcription (termed Site C); this site contains sequences required for general transcription in virtually all tissues that express PEPCK-C. A second site was at +1900 bp, within intron 4 (termed Site K); hypersensitive Site K is unique to kidney tubules and was not found in a line of cultured kidney cells that did not express the PEPCK-C gene. Support for the importance of the kidney-specific hypersensitive sites for renal PEPCK-C gene expression can be found in studies with transgenic mice in which the tissue-specific regulation of the gene promoter was studied. Transgenes that included segments of the PEPCK-C gene promoter ranging from −2000 to +73 or −109 to +73 bp linked to a marker gene were expressed in the appropriate mouse tissues (i.e. liver and adipose tissue) (24Patel Y.M. Yun J.S. Liu J. McGrane M.M. Hanson R.W. J. Biol. Chem. 1994; 269: 5619-5628Abstract Full Text PDF PubMed Google Scholar). However, expression of the transgene in kidney was only partially induced compared with a transgene containing the entire PEPCK-C gene (25Eisenberger C.L. Nechushtan H. Cohen H. Shani M. Reshef L. Mol. Cell. Biol. 1992; 12: 1396-1403Crossref PubMed Scopus (48) Google Scholar). These findings suggest that the kidney-specific DNase I-hypersensitive site at +1900 bp (Site K in intron 4) is required for the full physiological level of expression of the gene in kidney cortex. The role of the sequences in Site K in the regulation of renal PEPCK-C gene transcription is not clear at this time. An analysis of the PEPCK-C gene promoter in transgenic mice indicated that a region between −540 and −362 bp is required for the glucocorticoid induction of PEPCK-C gene transcription in kidney (25Eisenberger C.L. Nechushtan H. Cohen H. Shani M. Reshef L. Mol. Cell. Biol. 1992; 12: 1396-1403Crossref PubMed Scopus (48) Google Scholar); this is consistent with the position of the GRU, which maps at −410 Site C contains a site (Fig. which is required for the renal specific transcription of the PEPCK-C gene and for its response to acidosis H. Olswang Y. K. Hanson R.W. Reshef L. PubMed Scopus Google Scholar). et al. (24Patel Y.M. Yun J.S. Liu J. McGrane M.M. Hanson R.W. J. Biol. Chem. 1994; 269: 5619-5628Abstract Full Text PDF PubMed Google Scholar) noted that the of this site in the PEPCK-C gene promoter in transgenic mice in of transcription from the promoter in kidney. As the region in hypersensitive Site C is not to the control of renal gene it is a key region for the expression of the PEPCK-C gene in all tissues studied to that binding at K and C to appropriate expression of the PEPCK-C gene in kidney cortex. DNase I-hypersensitive Site which maps at −4800 bp of the PEPCK-C can function to increase both the and of transcription of a gene that has been into transgenic mice Ip T. Y. Chalkley R. 1992; PubMed Scopus Google Scholar). In this it of a and the hypersensitive sites the region of the gene D. J. 10: PubMed Scopus Google Scholar, D.R. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar) and the gene D.R. Wilson D. Cell. 1989; Full Text PDF PubMed Scopus Google Scholar). Site A contains a sequence that a of the K. and L. As in detail this region is in the and It is that significant of sequence in the PEPCK-C gene and surrounding are in several that the level of sequence noted in areas that are of the region of the PEPCK-C gene for the importance of these of in the control of PEPCK-C gene In Fig. present the rat PEPCK-C gene and it with the sequence of the gene from other and The rat PEPCK-C gene has and the is virtually in the five mammalian studied and in but the first is not found in the and PEPCK-C genes. There is a region in intron of the PEPCK-C gene that is found in the five mammalian and in the regulatory of this region of the PEPCK-C gene is not The relative of the DNase I-hypersensitive sites are at the of Fig. and D are in the sequences that in location to Site A (at about −4800 Site B Site and Site E (at about bp) are not in as However, Site A is to a sequence at This site is present within the region to the start site of PEPCK-C gene in all five of the mammalian The of the DNase I-hypersensitive sites were on of the of in which is to it is possible that Site A to the site at bp noted in the analysis in Fig. it is a of the importance of this site as a that is involved in the development of PEPCK-C gene as by Ip et al. (20Ip Y.T. Granner D.K. Chalkley R. Mol. Cell. Biol. 1989; 9: 1289-1297Crossref PubMed Scopus (36) Google Scholar). Site which is at −1100 bp in most likely to a site at bp that is present in all mammalian in Fig. As this region contains a PPARγ2-binding that is required for the adipose tissue transcription of the mouse PEPCK-C gene. Finally, the of the PEPCK-C gene contains a region at bp that is in the five mammalian studied. This site most likely to Site which Ip et al. (20Ip Y.T. Granner D.K. Chalkley R. Mol. Cell. Biol. 1989; 9: 1289-1297Crossref PubMed Scopus (36) Google Scholar) mapped at +6300 have a of the region of PEPCK-C genes from a of ranging from to (Fig. the first bp of sequence to the start site of gene there are a of sequence between mammalian that directly to known transcription factor-binding sites within the rat PEPCK-C gene promoter. The PEPCK-C gene promoter with other mammals (Fig. 2B) a region of sequence to the transcription start site, but with little Several are from this example, the cAMP response of the PEPCK-C gene promoter is to an site that between a site and the cAMP response this site is present in the and A previously site was noted at bp of the gene promoter in these have shown that in both of these sites and protein transcription from the PEPCK-C gene promoter. and R. W. The site in the promoter is within previously to be a single regulatory W.J. Hanson R.W. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, H. Olswang Y. Nechushtan H. Hanson R.W. Reshef L. 1997; PubMed Scopus (32) Google Scholar); this is in all have analyzed (Fig. Finally, a site at to bp was identified by sequence demonstrated in studies that binding to this site is to induced by J. E. Liu D. Hakimi P. Hanson R.W. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). In to the there is the and position of the site in the PEPCK-C gene this transcription factor is involved in the response of the PEPCK-C gene to insulin K. P. D. Hakimi P. M. P. Hanson R.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. D. Hanson R.W. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). with and the response of the hepatic PEPCK-C gene to insulin. This response is critical for the regulation of hepatic glucose and for glyceroneogenesis in liver. It is thus of that the PEPCK-C gene promoter has several regulatory elements with with the site in the low gene promoter K. D. Hanson R.W. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). These elements were shown by to to In contrast, the rat PEPCK-C gene promoter the protein to K. D. Hanson R.W. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the other the mouse PEPCK-C gene promoter not have a site on the sequence of these As in detail there is a region at about bp in the PEPCK-C gene promoter that is found in the of all mammalian analyzed (i.e. and but is not present in and (Fig. 2A). This region contains a PPARγ2-binding site that transcription of the PEPCK-C gene in adipose tissue (22Devine J.H. Eubank D.W. Clouthier D.E. Tontonoz P. Spiegelman B.M. Hammer R.E. Beale E.G. J. Biol. Chem. 1999; 274: 13604-13612Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and the of the in liver H. Kochan K. Chakravarty K. Cohen H. Blum B. Olswang Y. Hakimi P. Xu C. Massillon D. Hanson R.W. Reshef L. J. Biol. Chem. 2005; 280: 33873-33884Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Thus, in glucocorticoids the of fatty between liver and adipose This involves a regulation of PEPCK-C gene transcription in the two tissues the in the gene promoter in adipose tissue and the in liver. The of this region in and suggests a different of transcriptional control in adipose tissue gene transcription from these in of specific transcription factors that to of the PEPCK-C gene promoter in to areas of there is little information on the mechanisms involved in the methylation status of the gene. factors that associated with the which regulatory to the as changes in and methylation are likely to a role in the control of PEPCK-C gene transcription. The in this minireview an from which to the mechanisms by which chromatin modification PEPCK-C gene transcription. are to for this in