NEGATIVE REGULATION OF THE VASCULAR SMOOTH-MUSCLE ALPHA-ACTIN GENE INFIBROBLASTS AND MYOBLASTS - DISRUPTION OF ENHANCER FUNCTION BY SEQUENCE-SPECIFIC SINGLE-STRANDED-DNA-BINDING PROTEINS

Transcriptional activation and repression of the vascular smooth muscl e (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximat ely 30-bp polypurine-polypyrimidine tract. This region contains bindin g sites for an essential transcription-activating protein, identified as transcriptional enhancer factor 1 (TEF-1), and two tissue-restricti ve, sequence-specific, single-stranded-DNA-binding activities termed V ACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-bind ing activity, while VACssBF1 and VACssBF2 have little, if any, affinit y for double-stranded DNA. Site-specific mutagenesis experiments demon strate that the determinants of VACssBF1 and VACssBF2 binding lie on o pposite strands of the DNA helix and include the TEF-1 recognition seq uence. Functional analysis of this region reveals that the CCAAT box-b inding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in act ivating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. I mportantly, replacement of the TEF-1-binding site with that for NF-Y d iminishes the ability of VACssBF1 and VACssBF2 to bind to separated si ngle strands. Additional activating mutations have been identified whi ch lie outside of the TEF-1-binding site but which also impair single- stranded-DNA-binding activity. These data support a model in which VAC ssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcrip tion by stabilizing a local single-stranded-DNA conformation, thus pre cluding double-stranded-DNA binding by the essential transcriptional a ctivator TEF-1.