Structural and functional analysis of the rat malic enzyme gene promoter.

Abstract

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences + 1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.Rat liver malic enzyme (ME) (EC 1.1.1.40) plays an important role in lipogenesis. We have previously reported cloning (22) and the sequence of ME mRNA (21) and shown its regulation by thyroid hormone and a high carbohydrate diet (4)(5)(6). To investigate regulation of the ME gene, we have identified and sequenced its 5'-flanking region.The sequence shown in Fig. 1 extends from the EcoRI site through the transcription start site and includes 109 base pairs (bp) of the first exon with a portion of the first intron. The structural organization of the ME promoter differs from that found in tissue-specific promoters, which are usually rapidly regulated and often switched off, but it resembles more closely promoter regions of several eucaryotic constitutive or "housekeeping" genes (23-28, 30, 31), the simian virus 40 late promoter (9), and some recently characterized proto-oncogenes (16,17). A TATA box (2), usually located 20 to 30 bp upstream from cap sites, lies at -622 ( Fig. 1). The sequence CCGAT, between -144 and -140, resembles the canonical CCAAT consensus sequence often found at a position 80 bp from transcription start sites in many eucaryotic promoters (7). The most striking motif is the nine hexanucleotides CCGCCC. Six of these GC boxes are located upstream from the major cap site from -376 to -10. One GC box is present in an untranslated region, while the last two GC boxes are found within the first intron. Noteworthy is the number of nucleotides, (i.e., 65, 63, and 61) separating the GC motifs at -46, -111, -174, and -235, respectively. This rather equal spacing, also seen in the 5'-flanking region of the adenosine deaminase gene (31), might play a role in the chromatin assembly required for expression. The 6-bp sequence of the GC motif is the same sequence that is repeated six times within the simian virus 40 promoter (9) and has been shown to be a core element in the decanucleotide sequence, exhibiting high affinity binding for Spl (1,8 with one deletion) to the consensus sequence of the 3' region of a 48-bp repeated element in the mouse dihydrofolate reductase gene (25).Farther downstream from -124 to -119 is the hexanucleotide sequence 5'-CGCTTC-3'. Three copies of this sequence occur within the boundaries of the Moloney murine sarcoma virus long terminal repeat distal transcription signal (11), immediately downstream from the CCAAT box. The position of this element relative to a CAT-like box (-144) found in the ME gene 5'-flanking region imitates the position identified in the viral long terminal repeat. We also note t...

Transcriptional Regulatory Patterns of the Myelin Basic Protein and Malic Enzyme Genes by the Thyroid Hormone Receptors α1 and β1

“…Plasmids-The construction of chloramphenicol acetyltransferase (CAT) reporter genes, MBP(Ϫ256/ϩ3)-CAT and ME(Ϫ315/ϩ3)-CAT, has been previously described (29,33), and these genes are here referred to as MBPϪ256 and MEϪ315, respectively. The MBP/ME TRE and the MBPϪ256/5Јm reporter gene were obtained by PCR mutagenesis of the MBPϪ256 reporter according to the protocol described for PCR technology (34).…”

Role of Sp1 in insulin regulation of gene expression

“…However, Sp1 has unique features that include the combination of transcriptional activation domains comprised of two glutamine-rich domains A and B, a weakly basic domain, and a carboxyl-terminal D domain ( Fig. 1; Kadonaga et al 1987, 1988, Courey & Tjian 1988, Pascal & Tjian 1991, Phillipsen & Suske 1999). Sp1 may form oligomers through interaction of domains A and B resulting in a tetrameric configuration.…”

“…The ME promoter is GC-rich and contains numerous Sp1 motifs (Moroika et al 1988, Garcia-Jimenez et al 1994. Transcription factor AP-1 has previously been shown to play a role in mediating insulin regulation of the ME promoter (Streeper et al 1998).…”

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

“…In addition to insulin, thyroid hormones and fatty acids are also positive regulators of ME, whereas glucagon, cAMP inducers, and retinoic acid inhibit its expression. The rat ME gene promoter lacks both a TATA box and CAAT sequence and displays an extremely GC-rich content in its proximal region (16). Functional response elements for thyroid hormone receptors (17) and peroxisome proliferator-activated receptors (18) have been located within the ME promoter.…”