Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter.
rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.
Sheng
Z H
ZH
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104-6059.
Zhang
H
H
Barchi
R L
RL
Kallen
R G
RG
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D007438:Introns; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009132:Muscles / Q000166:cytology; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D015222:Sodium Channels / Q000235:genetics*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015222:Sodium Channels