Sp Family Members and Nuclear Factor-Y Cooperatively Stimulate Transcription from the Rat Pyruvate Kinase M Gene Distal Promoter Region via Their Direct Interactions

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (ϳ2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.

Section: Discussionmentioning

confidence: 99%

Transcriptional Regulation of the Murine Acetyl-CoA Synthetase 1 Gene through Multiple Clustered Binding Sites for Sterol Regulatory Element-binding Proteins and a Single Neighboring Site for Sp1

Ikeda

Yamamoto

Okamura

et al. 2001

“…Moreover, there are abundant examples of promoters that require the concerted action of Sp1 and NF-Y (see Refs. 38 -42 for recent studies), and in two cases a physical interaction between these factors has been demonstrated (43,44). In addition, other positive elements, located between Ϫ63 and ϩ66 may be required for optimal transcription, given that constructs p39␤4LUC, p63␤4LUC (Fig.…”

Section: Discussionmentioning

confidence: 99%

Transcription Factors NF-Y and Sp1 Are Important Determinants of the Promoter Activity of the Bovine and Human Neuronal Nicotinic Receptor β4 Subunit Genes

Valor

Campos‐Caro

Carrasco-Serrano

et al. 2002

The ␤4 subunit is a component of the neuronal nicotinic acetylcholine receptors which control catecholamine secretion in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was characterized. A proximal region (from ؊99 to ؊64) was responsible for the transcriptional activity observed in chromaffin, C2C12, and COS cells. Within this region two cis-acting elements that bind transcription factors Sp1 and NF-Y were identified. Mutagenesis of the two elements indicated that they cooperate for the basal transcription activity of the promoter. The human ␤4 promoter, that was also characterized, shared structural and functional homologies with the bovine promoter. Thus, two adjacent binding elements for Sp1 and NF-Y were detected. Whereas the Sp1 site was an important determinant of the promoter activity, the NF-Y site may have cell-specific effects. Given that these promoters showed no structural or functional homology with the previously characterized rat ␤4 subunit promoter (Bigger, C. B., Casanova, E. A., and Gardner, P. D. (1996) J. Biol. Chem. 271, 32842-32848) except for the involvement of an Sp1 binding element, we propose that constitutive expression of the ␤4 subunit gene in these three close species may be controlled by the general transcription factor Sp1. Nevertheless, other components could determine species-specific ␤4 subunit expression. Cloning of nicotinic acetylcholine receptor (nAChRs)1 subunit cDNAs has revealed that the molecular heterogeneity of the gene families encoding the different receptor subunits is responsible for the pharmacological and functional diversity of nAChRs in the peripheral and central nervous systems (1, 2). The varied tissue-, region-, and development-specific distribution of nAChRs subunits (3) has suggested that complex transcriptional mechanisms direct nAChR expression. Moreover, potential changes in subunit transcription in response to modulation of synaptic function, might have important consequences on the signals transduced by nAChRs (4, 5). For these reasons considerable effort has been dedicated to the elucidation of the molecular basis for the transcriptional regulation of neuronal nAChRs, and thus several cis-and trans-acting elements in the promoter of the different nAChRs subunits have been identified (6).In our laboratory we have previously isolated and characterized the promoters of the bovine ␣5 (7) and ␣7 (8) subunits. These subunits are expressed in the chromaffin cells of the adrenal gland composing two different receptor subtypes, one of them formed by ␣7 subunits (9) and the other by ␣3, ␤4, and ␣5 subunits (10). Interestingly, the genes of the latter subunits are clustered in the vertebrate genome (11, 12) and may have common patterns of regulation (13). We have analyzed here the bovine and human ␤4 promoters, finding that they are highly homologous in their proximal regions as well as in the ciselements governing basal transcriptional activity. Although their sequences differ from the one of the rat ␤4 promoter (14...

“…All plasmids used for transfection were prepared using the Qiagen plasmid Midi kit. The transfection conditions and luciferase reporter assays have been described previously (33).…”

Section: Methodsmentioning

confidence: 99%

“…Histidine-tagged NF1-C was expressed from pQE-NF1-C in E. coli. Preparation of GST fusion proteins containing NF1 family members or HNF1␣ and pull-down analysis were carried out as described previously (33). Finally, samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography or Western blotting.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Nuclear Factor 1 Family Members Interact with Hepatocyte Nuclear Factor 1α to Synergistically Activate L-type Pyruvate Kinase Gene Transcription

Satoh

Noaki

Ishigure

et al. 2005

Self Cite

Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cisregulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1␣ binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5-flanking region up to ؊189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1␣, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1␣-binding site, raising the possibility that NF1 proteins function as HNF1␣ co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1␣ bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1␣ via their DNA-binding domains. This interaction enhanced HNF1␣ binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1␣ plays an important role in hepatic L-PK transcription.Pyruvate kinase (ATP, pyruvate 2-O-phosphotransferase; PK) 2 is an important regulatory enzyme in the glycolytic pathway. Four PK isozymes are known to exist in mammals and are designated as L, R, M 1 , and M 2 types (1). They are produced from two genes, PKL and PKM, by alternative use of two promoters and mutually exclusive alternative splicing, respectively (2-4). The L-type isozyme (L-PK) is expressed in a tissue-specific manner; this form is expressed primarily in hepatic parenchymal cells and is also present in pancreatic ␤ cells, kidney, and small intestine (1). Hepatic L-PK gene expression is also transcriptionally controlled positively by glucose in the presence of insulin and negatively by polyunsaturated fatty acids or glucagon via cyclic AMP (5-8). These regulations of the L-PK gene transcription are mediated via the multiple cis-regulatory elements located within a 170-bp upstream region from the transcription start site, as shown in Fig. 1 (8 -10). We have named these elements L-I (Ϫ94 to Ϫ76), L-II (Ϫ149 to Ϫ126), and L-III (Ϫ170 to Ϫ150) (9). In addition, another group has identified a weak negative element (Ϫ116 to Ϫ99) between L-I and L-II (10). Although this element has been named L2, we will refer to this as L-IIA and L-II as L-IIB hereafter. Although L-IIB is considered to be a regulatory element...