The Roles of Sterol Regulatory Element-binding Proteins in the Transactivation of the Rat ATP Citrate-Lyase Promoter

Abstract

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from ؊114 to ؊60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the ؊102/؊94 region, immediately upstream of the inverted Y-box, and the ؊70/؊61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter. ATP citrate-lyase (ACL)1 is a cytosolic enzyme that catalyzes the cleavage of citrate into oxaloacetate and acetyl-CoA (1). In liver and adipose tissue, this enzyme plays an important role in supplying acetyl-CoA for both fatty acid and cholesterol synthesis (2). As the specific inhibition of ACL in rats significantly decreases the plasma levels of triacylglycerol and cholesterol, ACL is expected to be a potential target for hypolipidemic intervention (3, 4). The activity of ACL is mainly regulated at the level of transcription by diet regimen and insulin, like other lipogenic enzymes, such as fatty acid synthase and acetyl-CoA carboxylase (5). The sequences of the 5Ј flanking region of the ACL gene are highly conserved in humans and rats, whereas there is no homology in the regions of the 5Ј untranslated region and the first intron, suggesting that transcription is regulated in the same manner in these two species (6, 7). Although ACL plays an important role in fatty acid and cholesterol biosynthesis and is highly controlled by diet at the transcription level, studies on the structure and function of this promoter have been very limited thus far.SREBPs are the transcription factors that regulate the transcription of ma...

Differential Regulation of Human and Mouse Orphan Nuclear Receptor Small Heterodimer Partner Promoter by Sterol Regulatory Element Binding Protein-1

“…The mechanism for the differences in sterol regulation between SREBP-1c and Ϫ2 is currently unknown; however, the relative low sequence homology between their cholesterol regulatory carboxyl-terminals might be involved (49). In addition, previous reports have suggested that SREBP-1 and -2 have different binding affinities to SREs in ATP citrate-lyase promoter (35). However, we could not observe any difference in the binding affinities of SREBP-1 and -2 to SRE1 within the hSHP promoter (data not shown).…”

“…Plasmids and DNA Construction-SREBP expression plasmids pCSA10, pCMVhSREBP-1c 436, pCS2, and p5xSRE-tk/Luc were described previously (35), and the cDNA encoding SREBP-1c/ADD1-403 for adenovirus recombinant was described previously (45). The wildtype and mutant reporter plasmids of the hSHP promoter were cloned into the pGL2-basic plasmid (Promega) using restriction enzymes and specific primers.…”

“…Recombinant SREBP-1 proteins (1, 2, and 4 g), purified as described in Ref. 35, were incubated with 3 ϫ 10 5 cpm of labeled probe for 20 min on ice under the condition of 10 mM HEPES, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 7% (v/v) glycerol, and 2 g of poly(dI-dC), and then 50 l of diluted DNase I solution (0.002-0.001 units/ l) was added to the DNA-protein binding reactions.…”

Development of ligand-dependent regulatory system and its application to gene therapy of insulin-dependent diabetes mellitus

“…ACL promoter activity was increased by the overexpression of SREBP-1a and SREBP-2. However, the ACL minimal promoter -60/-67 shows very low basal activity and did not respond to SREBPs (Moon et al, 2000). ACL minimal promoter -60/+67 drastically elevated the ligand-induced expression levels, which were 16 and 25 folds higher than those of artificial TATA promoter.…”

“…SREBP-1a AD was obtained from pET-SREBP-1a (Moon et al, 2000) by BamHI/SacI digestion. SREBP-2 AD was generated by PCR with primer set of 5'-GGCGAATTCAGAT-CCATGGACGACAGCGGC-3', 5'-CTGCTAGATCTG-CTGCCACTGCTGCC-3' from pCS2 (Moon et al, 2000), and then digested with EcoRI/BglII. VP16 AD was prepared from pACT (Stratagene) by digestion with EcoRI/BglII.…”