Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.
Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.
Sun
S
S
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Stoflet
E S
ES
Cogan
J G
JG
Strauch
A R
AR
Getz
M J
MJ
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D000199:Actins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000091262:TEA Domain Transcription Factors; C083215:Tead1 protein, mouse; D014157:Transcription Factors