In mammals there are at least three isoforms of the glycolytic enzyme enolase encoded by three similar genes:a, p and y. In this report we describe the isolation and characterization of the human a-enolase locus. The gene appears to exist as a single copy in the haploid genome and is composed of 12 exons distributed over more than 18000 bases. The structure of this gene has a high degree of similarity to that of the human and rat y-enolase genes, with identical positions for all the intron regions. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites. The putative promoter region, like that of other housekeeping genes, lacks canonical TATA and CAAT boxes, is extremely G + C-rich and contains several potential SP1 binding sites. Furthermore, various sequences similar to known regulatory elements were detected.The functional role of multiple enzyme isoforms and the regulatory mechanisms required for developmental and tissuespecific expression of these isoforms are still largely unknown. The glycolytic enzyme enolase (2-phospo-~-glycerate hydrolase) represents a suitable model for the study of these mechanisms. Three distinct isoforms of the enzyme, referred to as a or non-neuronal enolase, p or muscle-specific enolase and y or neuron-specific enolase are present in both avian and mammalian tissues [l]. The active enzyme is a homodimer of non-covalently linked subunits, each a, p and y subunit is encoded by a separate gene as previously proposed [2] and recently established by cDNA cloning in human [3-51, rat [6 -81, and mouse [9].While the y isoform is mainly detected in cells of neuronal origin and the p isoform is found in adult skeletal muscle, the a isoform is widely distributed among different tissues and is the major form of enolase present in the early stage of embryonic development. Isoform switch occurs along with terminal differentiation in neurons and skeletal muscle cells from the a-to the y-and p-enolase respectively [2, 10, 111. Although the isolated isoenzymes have been studied for many years and a substantial body of information on their biochemical, kinetic, and immunological properties [I21 as well as on the crystallographic structure [I31 has been collected, very little is known about the control of expression of the enolase genes. Beside the already mentioned differential expression during development and in various cell types, transcriptional induction of the a-enolase gene has been shown upon mitogenic stimulation of human peripheral-blood lymphocytes [3], as well as in quiescent rat fibroblast stimulated with growth factors or serum [14]. High levels of expression of the y isoform have been detected in many tumors of nonneuronal origin [5] suggesting lack of tissue-specific control in transformed cells. Furthermore, the finding that one of the two enolase isoenzymes of the budding yeast Saccharomyces cerevisiue may be involved in both thermal tolerance and growth control [15] and the identification of the lens structural prote...
To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue-and stage-specific expression of the  enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3-proximal portion of the first intron (nucleotides ؉504 to ؉637) and acts as an enhancer irrespective of orientation and position from the homologous  enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the  enolase gene in skeletal muscle cells.
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