Molecular and functional analysis of the muscle-specific promoter region of the Duchenne muscular dystrophy gene.
Duchenne muscular dystrophy (DMD) gene transcripts are most abundant in normal skeletal and cardiac muscle and accumulate as normal myoblasts differentiate into multinucleated myotubes. In this report we describe our initial studies aimed at defining the cis-acting sequences and trans-acting factors involved in the myogenic regulation of DMD gene transcription. A cosmid clone containing the first exon of the DMD gene has been isolated, and sequences lying upstream of exon 1 were analyzed for homologies to other muscle-specific gene promoters and for their ability to direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs. The results indicate that the transcriptional start site for this gene lies 37 base pairs (bp) upstream of the 5' end of the published cDNA sequence and that 850 bp of upstream sequence can direct CAT gene expression in a muscle-specific manner. Sequence analysis indicates that in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. Our observation that only 149 bp of upstream sequence is required for muscle-specific expression of a chimeric CAT gene construct further implicates the CArG and myocyte-specific enhancer-binding nuclear factor 1 binding homologies as important domains in the regulation of this gene. On the other hand, the unique profile of myogenic cell line-specific induction displayed by our DMD promoter-CAT gene constructs suggests that other as yet undefined cis-acting sequences and/or trans-acting factors may also be involved.
Genetics Department, Hospital for Sick Children, Toronto, Ontario, Canada.
H J
HJ
Klamut
S B
SB
Gangopadhyay
R G
RG
Worton
P N
PN
Ray
D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D016189:Dystrophin; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009132:Muscles / Q000166:cytology / Q000502:physiology*; D009136:Muscular Dystrophies / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D016189:Dystrophin; D009124:Muscle Proteins; D012333:RNA, Messenger