The long terminal repeat (LTR) of ERV-9 human endogenous retrovirus binds to NF-Y in the assembly of an active LTR enhancer complex NF-Y/MZF1/GATA-2

J Biol Chem. 2005 Oct 21;280(42):35184-94. doi: 10.1074/jbc.M508138200. Epub 2005 Aug 16.

Abstract

The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.

Publication types

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

MeSH terms

  • Amino Acid Motifs
  • Base Sequence
  • Blotting, Western
  • CCAAT-Binding Factor / chemistry*
  • Cell Line, Tumor
  • Cell Nucleus / metabolism
  • Chromatin / chemistry
  • Chromatin Immunoprecipitation
  • DNA / chemistry
  • DNA-Binding Proteins / chemistry*
  • Deoxyribonuclease I / metabolism
  • Endogenous Retroviruses / genetics*
  • Enhancer Elements, Genetic
  • GATA1 Transcription Factor
  • GATA2 Transcription Factor / chemistry*
  • Green Fluorescent Proteins / metabolism
  • Humans
  • K562 Cells
  • Kruppel-Like Transcription Factors
  • Molecular Sequence Data
  • Mutation
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Protein Binding
  • Protein Structure, Tertiary
  • Sp1 Transcription Factor / metabolism
  • Terminal Repeat Sequences*
  • Transcription Factors / chemistry*
  • Transcription, Genetic
  • Transfection

Substances

  • CCAAT-Binding Factor
  • Chromatin
  • DNA-Binding Proteins
  • GATA1 Transcription Factor
  • GATA1 protein, human
  • GATA2 Transcription Factor
  • GATA2 protein, human
  • Kruppel-Like Transcription Factors
  • MZF1 protein, human
  • Sp1 Transcription Factor
  • Transcription Factors
  • Green Fluorescent Proteins
  • DNA
  • Deoxyribonuclease I