Transcription of the Human β Enolase Gene (ENO-3) Is Regulated by an Intronic Muscle-Specific Enhancer That Binds Myocyte-Specific Enhancer Factor 2 Proteins and Ubiquitous G-Rich-Box Binding Factors

Abstract

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.

Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human AβH-J-J Locus, Encoding Aspartyl-β-Hydroxylase, Junctin, and Junctate

“…In addition, there was diminished DNA binding activity of the muscle-specific transcription factor MEF-2. These data and other experimental evidence indicate that MEF-2 is involved in the activation of muscle-specific genes, including rat myosin light chain 2 slow, human myoglobin, rat muscle creatine kinase, mouse troponin C slow, and human beta enolase (14,15,20,26,50). In addition, MEF-2 has been proposed to serve as a nodal point in the mechanisms by which motor nerves control the program of gene expression in skeletal muscle fibers and in the pathway leading to cardiac hypertrophy.…”

The PVT‐1 oncogene is a Myc protein target that is overexpressed in transformed cells

“…Briefly, total cell lysates (30 mg) obtained from LAN-5 cells by extraction in RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0,5% DOC, 0.1% SDS) containing a protease inhibitor mixture (Roche), or nuclear extracts from RAT1-MycER cells (10 mg), prepared as described previously (Feo et al, 1995), were resolved by electrophoresis on SDS-polyacrylamide gel and electroblotted to a nitrocellulose membrane (Hybond-C, Amersham Bioscience). The membrane was incubated with affinity purified antibodies against c-Myc, N-Myc (Santa Cruz, Cat.…”

“…Cell extracts were prepared 48 h after transfection and Luciferase activity was measured in duplicate for all samples in a Turner 20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA) using the Promega luciferase assay system. Betagalactosidase activity was assayed as described previously (Feo et al, 1995). The ratio of luciferase activity to b-galactosidase activity in each sample served as a measure of the normalized luciferase activity.…”

Regulation of Murine Cytochrome c Oxidase Vb Gene Expression during Myogenesis

“…ZBP-89 also plays a critical role in the developmentally regulated expression of the gastrin gene in embryonic pancreas and adult stomach (33). The protein factor contains a transcription activation domain at its C terminus and a repressor domain at its N terminus (21), although the factor is more commonly known as a repressor (45). It is known to stabilize the binding of p53 to some critical target genes, resulting in arrest of cell growth (46).…”