Binding of phosphorylated Sp1 protein to tandem Sp1 binding sites regulates alpha2 integrin gene core promoter activity

Blood. 1997 Jul 15;90(2):678-89.


The alpha2beta1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the alpha2beta1, integrin in cells with megakaryocytic differentiation, we characterized the 5' flanking region of the alpha2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5' flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the alpha2 integrin gene located between bp -30 and -92 that is required for transcriptional activity of the alpha2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp -30 to bp -92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5', the 3', or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5' Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5' and the 3' Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, CD / biosynthesis*
  • Antigens, CD / genetics
  • Base Sequence
  • Binding Sites
  • Cell Differentiation
  • Consensus Sequence
  • DNA Footprinting
  • Enhancer Elements, Genetic
  • Humans
  • Integrin alpha2
  • Integrins / biosynthesis
  • Integrins / genetics
  • Megakaryocytes / cytology*
  • Megakaryocytes / physiology
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / metabolism
  • Sp1 Transcription Factor / biosynthesis
  • Sp1 Transcription Factor / metabolism*
  • Transcription, Genetic*
  • Transfection
  • Tumor Cells, Cultured


  • Antigens, CD
  • Integrin alpha2
  • Integrins
  • Nuclear Proteins
  • Recombinant Proteins
  • Sp1 Transcription Factor