Open AccessPromoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.
Author(s) -
Thomas Braun,
Egbert Tannich,
Gregor Buschhausen-Denker,
Hans Henning Arnold
Publication year - 1989
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.9.6.2513
Subject(s) - biology , footprinting , tata box , dna footprinting , transcription (linguistics) , transcription factor , myosin , microbiology and biotechnology , promoter , regulatory sequence , myogenesis , nuclear protein , dna binding protein , deoxyribonuclease i , binding site , myosin light chain kinase , gene , gene expression , genetics , linguistics , philosophy , base sequence
A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.