A muscle-specific enhancer within intron 1 of the human dystrophin gene is functionally dependent on single MEF-1/E box and MEF-2/AT-rich sequence motifs.
In previous studies we have described a 5.0 kb Hin dIII fragment downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity. Functional studies indicated that this enhancer is active in pre- and post-differentiated H9C2(2-1) myoblasts but functions poorly in L6 and C2C12 myotubes. The core enhancer region was delimited to a 195 bp Spe I- Acc I fragment and sequence analysis identified three MEF-1/E box and two MEF-2/AT-rich motifs as potential muscle-specific regulatory domains. EMSA competition and DNase footprinting indicated that sequences within a 30 bp region containing single adjoining MEF-1/E box and MEF-2/AT-rich motifs are target binding sites for trans -acting factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myotubes. Site-specific mutations within these motifs resulted in a significant reduction in enhancer activity in H9C2(2-1) myotubes. These results suggest that the mechanisms governing DMD gene expression in muscle are similar to those identified in other muscle-specific genes. However, the myogenic profile of enhancer activity and trans -acting factor binding suggests a more specialized role for this enhancer that is consistent with its potential involvement in dystrophin gene regulation in cardiac muscle.
Klamut
H J
HJ
Division of Experimental Therapeutics, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada. hklamut@oci.utoronto.ca
Bosnoyan-Collins
L O
LO
Worton
R G
RG
Ray
P N
PN
D000225:Adenine; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002531:Cerebellum / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016189:Dystrophin / Q000096:biosynthesis* / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D064326:MEF2 Transcription Factors; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D017570:MyoD Protein / Q000378:metabolism*; D018006:Myogenic Regulatory Factors; D016133:Polymerase Chain Reaction; D011689:Purkinje Cells / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013941:Thymine; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D016189:Dystrophin; D064326:MEF2 Transcription Factors; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D018006:Myogenic Regulatory Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D000225:Adenine; D013941:Thymine