Molecular Cloning and Functional Characterization of the Human Cytosolic Malic Enzyme Promoter: Thyroid Hormone Responsiveness

Abstract

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.

Using DNA microarray analyses to elucidate the effects of genistein in androgen‐responsive prostate cancer cells: Identification of novel targets

“…TaqMan reactions were carried out with the TaqMan universal PCR master mix (Applied Biosystems, Branchburg, NJ) in a total volume of 25 mL on an ABI-PRISM 7000 Sequence Detector (Applied Biosystems). Oligonucleotide primers and the TaqMan probes for SPAK [27] and ME1 [28] mRNAs were designed according to published sequences with the Primer Express 2.0 program (Applied Biosystems). Primers and probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PSA, and CDNK1A mRNAs were purchased from Applied Biosystems.…”

The adipose conversion process: regulation by extracellular and intracellular factors

“…GonzalezManchon et al [84] have suggested that, in the absence of T 3 , binding of T 3 receptors homodimers to T 3 RE represses human malic enzyme gene expression. These homodimers would dissociate from the T 3 RE when T 3 is present and the receptors could then heterodimerize with RXRα, this new complex activating malic enzyme gene expression.…”

Cloning and functional characterization of the ovine malic enzyme promoter

“…Ovine and human sequences show 86% identity in the region −224/−92, 84% identity in the region −744/−682 and 87% identity in the region −1126/−1075. The region corresponding to the major transcription start site of the human ME1 gene (González-Manchón et al, 1997) is also conserved in the ovine sequence (Fig. 1).…”

“…As in human, rat and chicken (Morioka et al, 1988;Hodnett et al, 1996;González-Manchón et al, 1997), the ovine ME1 promoter contains no TATA-box upstream of the transcription start site. Similar to the other species, the region proximal to the translation start site has a high GC content (76% from −473 to −1).…”

“…Refeeding after caloric restriction results in an increased ME1 gene expression in the liver and adipose tissue (Stelmanska et al, 2004). The sequences of the 5′-flanking regulatory regions have been functionally characterized for the ME1 genes of rat (Morioka et al, 1988), chicken (Hodnett et al, 1996) and human (González-Manchón et al, 1997).…”