Transcription of the human beta 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.
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 beta 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 beta 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 beta enolase gene in skeletal muscle cells.
Feo
S
S
Istituto Biologia dello Sviluppo del Consiglio Nazionale delle Richerche, Palermo, Italy.
Antona
V
V
Barbieri
G
G
Passantino
R
R
Calì
L
L
Giallongo
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D006801:Humans; D007438:Introns; D064326:MEF2 Transcription Factors; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D018006:Myogenic Regulatory Factors; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010751:Phosphopyruvate Hydratase / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D010751:Phosphopyruvate Hydratase