Characterization of the GArC motif. A novel cis-acting element of the human cardiac myosin heavy chain genes.
A positive element between positions -924 and -851 and a negative element between -851 and -762 of the 5'-upstream region of the alpha-myosin heavy chain gene were identified through transient transfection assays in primary cultures of neonatal rat heart cells. Subsequent DNase I protection analysis revealed almost identical footprints at two positions (GAAAAATCT at -904 to -896 and GAAAATCT at -823 to -816). We have designated this sequence the GArC motif (for G,AT-rich,C). Gel mobility shift assays demonstrated the formation of specific complexes with GArC oligomers when either rat heart, rat liver, or HeLa cell nuclear extracts were used. Competition studies with unlabeled GArC oligomers resulted in a loss of binding. Oligomers were also made to the Xenopus cytoskeletal actin serum response element and to a segment of the alpha-MyHC gene (AT-core), each with a similar AT-rich core sequence. No detectable loss of binding resulted from the addition of an excess of either of these unlabeled oligomers. Southwestern blot analysis identified several proteins which interacted with the GArC element, suggesting the presence of a group of related trans-acting factors. Analysis of a sequence in the beta-MyHC gene with the same AT-rich core was negative, suggesting a role for the bases surrounding the protected area in binding. We propose that the GArC motif, together with its associated trans-acting factor(s), provides a novel mechanism of transcriptional control in addition to those previously reported for the cardiac myosin heavy chain genes.
Mably
J D
JD
Toronto Hospital, Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
Sole
M J
MJ
Liew
C C
CC
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I; D006367:HeLa Cells; D006321:Heart / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D009218:Myosins / Q000235:genetics*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D015345:Oligonucleotide Probes; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014162:Transfection
D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I; D009218:Myosins