Malic Enzyme Promoter Research Articles

Cloning and functional characterization of the ovine malic enzyme promoter

While in human and rodents lipogenesis occurs predominantly in the liver, adipose tissue is the major site in ruminants. Here we report the nucleotide sequence of the 5′-flanking region of the ovine malic enzyme gene ( ME1). The ME1 promoter is located within a GC-rich region fulfilling the criteria of CpG islands and lacks a TATA-box. Deletion analysis identified a region (−231/−170) that suppressed promoter activity in luciferase assays in HepG2 hepatoma cells but not in 3T3-L1 adipocytes. This region contains a putative triiodothyronine response element (T3RE) that differs from the human ME1 T3RE by two nucleotides. When the human ME1 T3RE was introduced into the ovine ME1 promoter context, transcriptional activity was increased in the hepatic cell lines HepG2 and H4IIE but not in differentiated 3T3-L1 cells. Our results suggest that the sequence of the T3RE in the ME1 promoter determines differences in the tissue/species activity of malic enzyme in ruminants and human.

Human Spot 14 protein is a p53-dependent transcriptional coactivator via the recruitment of thyroid receptor and Zac1

Spot 14 is an acidic homodimeric protein with no sequence similarity to other mammalian gene products. Its biochemical function remains unclear. Recent studies have shown that the human Spot 14 locus is in the chromosomal region 11q13 and is frequently amplified in breast cancers, suggesting that it plays a role in the gene regulation involved in cell growth during tumorigenesis. Our previous work has demonstrated that human Spot 14 protein physically and functionally interacts with thyroid receptor in the regulation of malic enzyme gene expression. In this study, we investigated the subcellular distribution of human Spot 14 protein using enhanced green fluorescence protein and infer that its localization might be affected by thyroid hormone. Our results also demonstrate that the potential transactivation activity of human Spot 14 protein is regulated by its C-terminal region. Human Spot 14 protein is involved both in the regulation of malic enzyme promoter activity, and in the regulation of p53-dependent transactivation. It does not interact directly with p53, whereas it is able to directly interact with thyroid receptor and Zac1 (zinc finger protein which regulates apoptosis and cell cycle arrest 1) using glutathione-S-transferase pull-down assay. Hence, human Spot 14 protein might regulate the p53 target gene, p21WAF1/Cip1, via its direct interaction with the thyroid receptor or other p53 coactivators, such as Zac1. These findings provide a molecular rationale for the role of human Spot 14 protein in the p53-dependent transcriptional activation of specific genes via diverse pathways in cells.

Alterations in retinoid X receptor-α expression contribute to cell-type dependent differences in thyroid hormone regulation of malic enzyme transcription

Triiodothyronine (T3) stimulates a marked increase (>40-fold) in transcription of the malic enzyme gene in chick embryo hepatocytes (CEH), but has no effect on malic enzyme transcription in chick embryo fibroblasts (CEF) that express nuclear T3 receptors (TR) at levels which are similar to those of CEH. Heterodimerization of the TR with other nuclear proteins is a potential mechanism for the regulation of T3 action. For example, heterodimers of retinoid X receptors (RXR) and TR bind to T3 response elements (T3RE) with higher affinity and modulate transcription more effectively than TR homodimers. In the present report, we investigated the role of RXR in mediating differences in T3 responsiveness of the malic enzyme gene between CEH and CEF. Data from gel mobility shift analyses demonstrated that endogenous TRs from CEH and CEF bind to the major T3RE of the malic enzyme gene primarily as heterodimers with RXRα or a protein highly related to RXRα. The total binding activity of RXRα/TR complexes in CEF was decreased relative to that observed in CEH. Cell-type dependent differences in RXRα/TR complex formation were greater in cells incubated in the presence of T3 because T3 treatment increased RXRα/TR binding activity in CEH but had no effect on RXRα/TR binding activity in CEF. Decreased RXRα/TR complex formation in CEF relative to CEH was associated with a reduction in the abundance of RXRα protein and RXRα mRNA in the former cell-type. Expression of exogenous RXRα in CEF increased the T3 responsiveness of the malic enzyme promoter by about 4-fold. In contrast, expression of exogenous RXRα in CEH had no effect on the regulation of malic enzyme transcription by T3. These observations support the hypothesis that alterations in RXRα expression contribute to cell-type dependent differences in T3 responsiveness of the malic enzyme gene.

Hexachlorobenzene, a dioxin-type compound, increases malic enzyme gene transcription through a mechanism involving the thyroid hormone response element.

Hexachlorobenzene (HCB) is a dioxin-type chemical that acts mainly through the aryl hydrocarbon receptor. Chronic exposure of rats to HCB increases the activity of malic enzyme (ME). In this report, we show that this increase is correlated with an induction of ME messenger RNA (mRNA) levels, with the maximal HCB effect achieved after 9 days of intoxication. This effect is specific for ME, as other liver enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase, and mitochondrial alpha-glycerol-3-phosphate dehydrogenase, are not affected by HCB. The induction of ME mRNA levels is accompanied by an increase in ME promoter activity, as demonstrated by transient transfection experiments performed in rat hepatoma H35 cells. In an attempt to identify the cis-regulatory elements responsible for the HCB effect, different promoter deletions and mutations were used. The results obtained localize the responsive region between positions -315 and -177. This region does not contain either consensus xenobiotic response or activating protein-1 elements, the two main mediators of dioxin compounds described to date. In contrast, a thyroid hormone response element (TRE) is located between -281 to -261. Deletions and mutations of the TRE element do not respond to HCB, demonstrating that this element mediates the response of this dioxin-type compound. As ME gene expression is regulated mainly by thyroid hormones, we next investigated the role of T3 receptor (T3R) in the ME gene transcriptional induction mediated by HCB. Using Scatchard analysis, we show that neither T3R binding features for its ligand nor alpha1 or beta1T3R mRNA levels are changed with the toxic. In gel shift assays, however, we observed that protein/DNA complexes formed on TRE from the ME promoter were induced by HCB. Using an oligonucleotide with a mutation that eliminates the TRE function, we demonstrate a loss of the induced protein/DNA complexes. Together, these data suggest that the dioxin-type compound HCB increases ME gene transcription by modulating the levels of still unidentified nuclear proteins that bind to the TRE element of the ME promoter.

Insulin-induced Early Growth Response Gene (Egr-1) Mediates a Short Term Repression of Rat Malic Enzyme Gene Transcription

In this report we have studied insulin regulation of malic enzyme (ME) gene transcription in rat H-35 hepatoma cells and localized the insulin-responsive region of the ME promoter between positions -177 and -102. This region contains a putative insulin response element (IRE-II). When nuclear extracts from untreated or insulin-treated H-35 cells were incubated with IRE-II, transcription factors Sp1 and Sp3 were observed to bind constitutively to this element, whereas insulin induces the quick and transient binding of an insulin response factor. This induction requires de novo protein synthesis. Competition and supershift assays demonstrated that the insulin response factor is the immediate-early gene Egr-1. In vitro assays revealed that Egr-1 displaces Sp1 from its binding site in IRE-II. Insulin induces Egr-1 mRNA, with a time course pattern that corresponds perfectly to the Egr-1 binding to IRE-II. This induction depends on the activation of mitogen-activated protein (MAP) kinase, and it is phosphatidylinositol 3-kinase-independent, as demonstrated with specific inhibitors for both pathways. By cotransfecting the wild-type or a dominant negative Ras, an upstream regulator of MAP kinase, we show that Ras inhibits ME promoter activity. Furthermore, overexpression of Egr-1 in H-35 cells represses the ME gene promoter in a dose-dependent manner. These results suggest that insulin induces a quick, transient, and Ras/MAP kinase-dependent activation of Egr-1 which leads to a transient repression of ME gene transcription. On a late phase, insulin would activate a different, Egr-1-independent pathway, which would result in activation of the ME gene.

Effect of the CCAAT/Enhancer Binding Protein on Expression of the Gene for Chicken Malic Enzyme

The gene for malic enzyme is expressed at a high level in chick embryo-hepatocytes (CEH) treated with triiodothyronine (T3) and at a low level in the absence of T3. In chick-embryo fibroblasts (CEF), expression of the malic enzyme gene is low and not regulated by T3. Specific nuclear proteins from both CEH and CEF bound to a consensus CCAAT/enhancer binding protein (C/EBP) site at −335 to −327 bp of the malic enzyme gene. The level of binding was much higher in extracts from CEH than in extracts of CEF, and the complexes formed had different mobilities. C/EBPα was present in the complex that bound to the C/EBP site in nuclear extracts from CEH but not in those from CEF. The C/EBP element was necessary and sufficient to bestow full T3 responsiveness to 5800 bp of 5′-flanking DNA of the malic enzyme gene in CEH. C/EBPα was not detectable in wild-type CEF, and deletion of the C/EBP binding site had no effect on expression of transgenes containing 5800 bp of 5′-flanking DNA of the malic enzyme gene. In CEF, overexpression of C/EBPα stimulated promoter activity of constructs that contained the C/EBP site linked to the malic enzyme promoter or a heterologous reporter. The results suggest that C/EBPα or a closely related isoform is involved in the tissue-specific expression of the malic enzyme gene.

A phorbol ester-insensitive AP-1 motif mediates the stimulatory effect of insulin on rat malic enzyme gene transcription.

In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyl-transferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.

A Phorbol Ester-Insensitive AP-1 Motif Mediates the Stimulatory Effect of Insulin on Rat Malic Enzyme Gene Transcription

In liver, insulin stimulates the transcription of the gene encoding the cytosolic form of malic enzyme (ME) and modulates protein binding to two putative insulin response sequences (IRSs) in the ME promoter. One of these IRSs resembles that identified in the phosphoenolpyruvate carboxykinase (PEPCK) gene, whereas the other resembles that defined in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. To assess the functional significance of these changes in protein binding, a series of truncated ME-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into rat H4IIE hepatoma cells. Deletion of the PEPCK-like IRS motif had no effect on the stimulation of CAT expression by insulin. Instead, the stimulatory effect of insulin was mediated through an AP-1 motif and an Egr-1 binding site that overlaps the GAPDH-like IRS motif. Both the ME AP-1 motif and the AP-1 motif identified in the collagenase-1 gene promoter were able to confer a stimulatory effect of insulin on the expression of a heterologous fusion gene, but surprisingly only the latter was able to confer a stimulatory effect of phorbol esters. Instead, the data suggest that AP-1 binds the ME AP-1 motif in an activated state such that phorbol ester treatment has no additional effect. The collagenase and ME AP-1 motifs were both shown to bind mainly Jun D and Fra-2, with similar affinities. However, the results of a proteolytic clipping bandshift assay suggest that these proteins bind the collagenase and ME AP-1 motifs in distinct conformations, which potentially explain the differences in phorbol ester signaling through these elements.

Cell-Specific Regulation of Transcription of the Malic Enzyme Gene: Characterization of cis-Acting Elements That Modulate Nuclear T3 Receptor Activity

Stimulation of malic enzyme transcription by triiodothyronine (T3) is robust (>60-fold) in chick embryo hepatocytes, weak (5-fold) in chick embryo fibroblasts that stably overexpress the nuclear T3 receptor-α, and still weaker (1-fold) in chick embryo fibroblasts which contain nuclear T3 receptor levels that are similar to those of chick embryo hepatocytes. Using DNase I hypersensitivity, functional transfection, andin vitroDNA-binding analyses, four cis-acting elements were identified in the malic enzyme 5′-flanking DNA that conferred differences in nuclear T3 receptor activity between chick embryo hepatocytes and chick embryo fibroblasts. These cell-specific regulatory elements are located at −3895/−3890, −3761/−3744, −3703/−3686, and −3474/−2715 bp and overlap with DNase I hypersensitive sites that are observed in chromatin of chick embryo hepatocytes. Each element enhances T3 responsiveness of the malic enzyme promoter in chick embryo hepatocytes but has no effect on T3 responsiveness in chick embryo fibroblasts. Three of the cell-specific regulatory elements flank a previously identified DNA fragment (−3889 to −3769 bp; Hodnettet al., Arch. Biochem. Biophys.334, 309–324, 1996) that contains one major and four minor T3 response elements. The cell-specific regulatory element at −3703/−3686 bp binds to the liver-enriched factor, CCAAT/enhancer-binding protein-α, whereas cell-specific regulatory elements at −3895/−3890 and −3761/−3744 bp bind proteins of unknown identity. While the cell-specific regulatory element at −3761/−3744 bp contains sequences that resemble binding sites for CCAAT/enhancer-binding protein, activator protein-1, cyclic AMP response element binding protein, and NF-1, none of these proteins appear to bind to this DNA fragment. These data suggest that cell-specific differences in T3 responsiveness of the malic enzyme gene are mediated in large part by nonreceptor proteins that augment the transcriptional activity of the nuclear T3 receptor in hepatocytes.

Active repression by thyroid hormone receptor splicing variant alpha2 requires specific regulatory elements in the context of native triiodothyronine-regulated gene promoters.

Structural requirements for the inhibitory action of thyroid hormone receptor splicing variant alpha2 (TR alpha2) on T3/TRbeta1-mediated transactivation were investigated in native promoters of two T3-regulated genes: the brain-specific myelin basic protein (MBP) and the housekeeping malic enzyme (ME). T3/TRbeta1 transactivation of MBP256-chloramphenicol acetyl transferase (CAT) and ME315-CAT constructs was inhibited and unaffected by TR alpha2, respectively. In electrophoretic mobility shift assays, TR alpha2 bound MBP-thyroid response element (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR alpha2 binding but not inhibition of T3/TRbeta1-mediated transactivation. In the context of the MBP promoter, replacement of MBP-TRE with ME-TRE or exchange of MBP TATA-like box with the ME GC-rich region spanning the transcription start site abolished TR alpha2 dominant negative action. Simultaneous introduction of both MBP-TRE and MBP TATA-like box in the context of ME promoter, however, triggered TR alpha2 inhibition of T3/TRbeta1 transactivation, indicating that these regulatory elements are necessary, but not individually sufficient, to mediate TR alpha2 dominant negative activity. Functional studies at low TR alpha2/TRbeta1 ratios revealed that binding to TRE facilitates TR alpha2 dominant negative action while prevention of DNA interaction by altering TR alpha2 P-box structure preserved TR alpha2 inhibitory effect, although with lower potency. In conclusion, the results suggest that, in native promoters of T3-regulated genes, a dual molecular mechanism, with DNA-binding dependent and DNA-binding independent components, underlies TR alpha2 dominant negative activity.

Polarity and Specific Sequence Requirements of Peroxisome Proliferator-activated Receptor (PPAR)/Retinoid X Receptor Heterodimer Binding to DNA: A FUNCTIONAL ANALYSIS OF THE MALIC ENZYME GENE PPAR RESPONSE ELEMENT

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.

Molecular cloning and functional characterization of the human cytosolic malic enzyme promoter: thyroid hormone responsiveness.

We report the structural and functional features of the 5'-flanking region of the human cytosolic malic enzyme (ME) gene. A 2.2-kb subclone, comprising 1.5 kb upstream of the translation initiation codon, the first exon, and 0.7 kb of flanking intronic region, was sequenced and mapped to chromosome 6. The proximal promoter region is rich in G + C, lacks TATA or CCAAT boxes, and shows multiple transcription start sites, the major one 106 nucleotides upstream the ATG codon. Sequences -59/-13 and -137/-103 conferred maximal promoter activity. Deletional analysis revealed the presence of two regions positively regulated by 3,5,3'-triiodo-L-thyronine (T3). The proximal region confers the strongest T3 inducibility to the human ME as well as to a heterologous promoter. Thyroid hormone receptor beta (TRbeta) binds to an inverted palindromic T3 response element (TRE) at position -105/-87 in a manner that is prevented by T3. Nuclear extracts or in vitro-translated retinoid acid receptor alpha (RXR alpha) shifted the TRbeta retarded band to slower-mobility complexes, which are unaffected by T3. In the absence of T3, overexpression of TRbeta repressed the ME promoter activity, most probably, through binding of TRbeta homodimers to the TRE. Thus, T3 seems to control ME transcription by inducing the dissociation of TRbeta homodimers and the functional activation of liganded heterodimers.

Nutritional regulation of nucleosomal structure at the chicken malic enzyme promoter in liver.

Transcription of the chicken malic enzyme gene in the liver is stimulated by feeding and inhibited by starvation. Concomitant with the increase in transcription caused by refeeding, chromatin structure around the transcription start site of the malic enzyme gene is modified in the liver. Digestion of chromatin in isolated nuclei with DNase I revealed four feeding-induced DNase I hypersensitive sites (-220, -170, -130 and -70 bp) near the malic enzyme promoter. Similarly, digestion of chromatin with restriction endonucleases detected enhanced cleavage within this region when birds were refed. Micrococcal nuclease detected the presence of nucleosomes over this region in the starved state, but not in the fed state. After food was withdrawn from fed birds, nucleosomes were reformed in this region within 6 h. The speed and magnitude of the changes in nucleosomal structure in this region suggest that they did not require DNA replication.

Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element.

We previously showed that the 5'-flanking region of the malic enzyme (ME) gene contains a cis-regulatory element (-281 to -261) that binds thyroid hormone receptors and confers triiodothyronine (T3) inducibility of transcription to the ME promoter (Petty, K.J., Desvergne, B., Mitsuhashi, T., and Nikodem, V. M. (1990) J. Biol. Chem. 265, 7395-7400). In this report, we have used deletion and mutation analyses of the ME thyroid hormone response element (TRE) to evaluate the roles of several subregions of TRE in T3 binding and transactivation. ME TRE was shown to act as an enhancer conferring T3 responsiveness to a heterologous promoter thymidine kinase. Although T3 treatment induced the promoter activity, the absence of hormone resulted in repression as measured by the level of chloramphenicol acetyltransferase expression in the NIH 3T3 transient expression system in the presence of overexpressed receptor. The degree of repression was similar to the degree of T3 induction observed for the same TRE mutants. Mutation and deletion analyses indicated that the functional TRE is comprised of discrete regions that are not contiguous, with a dominant role of a cluster of G residues and an AGGACA sequence. Both functions, induction and repression of transcription, correlated with receptor binding to the ME TRE as determined by competition binding assays using wild type and mutated TRE as competitors.