title: Functional interaction of CP2 with GATA-1 in the regulation of erythroid promoters.
abstract: We observed that binding sites for the ubiquitously expressed transcription factor CP2 were present in regulatory regions of multiple erythroid genes. In these regions, the CP2 binding site was adjacent to a site for the erythroid factor GATA-1. Using three such regulatory regions (from genes encoding the transcription factors GATA-1, EKLF, and p45 NF-E2), we demonstrated the functional importance of the adjacent CP2/GATA-1 sites. In particular, CP2 binds to the GATA-1 HS2 enhancer, generating a ternary complex with GATA-1 and DNA. Mutations in the CP2 consensus greatly impaired HS2 activity in transient transfection assays with K562 cells. Similar results were obtained by transfection of EKLF and p45 NF-E2 mutant constructs. Chromatin immunoprecipitation with K562 cells showed that CP2 binds in vivo to all three regulatory elements and that both GATA-1 and CP2 were present on the same GATA-1 and EKLF regulatory elements. Adjacent CP2/GATA-1 sites may represent a novel module for erythroid expression of a number of genes. Additionally, coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated a physical interaction between GATA-1 and CP2. This may contribute to the functional cooperation between these factors and provide an explanation for the important role of ubiquitous CP2 in the regulation of erythroid genes.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, P.za della Scienza 2, 20126 Milano, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bosè
authors.forename: Cristina
authors.initials: C
authors.lastname: Fugazza
authors.forename: Maura
authors.initials: M
authors.lastname: Casalgrandi
authors.forename: Alessia
authors.initials: A
authors.lastname: Capelli
authors.forename: John M
authors.initials: JM
authors.lastname: Cunningham
authors.forename: Quan
authors.initials: Q
authors.lastname: Zhao
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Jane
authors.forename: Sergio
authors.initials: S
authors.lastname: Ottolenghi
authors.forename: Antonella
authors.initials: A
authors.lastname: Ronchi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D050982:GATA1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D050984:NF-E2 Transcription Factor, p45 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D050982:GATA1 Transcription Factor; D051741:Kruppel-Like Transcription Factors; D050984:NF-E2 Transcription Factor, p45 Subunit; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C428128:TFCP2L1 protein, human; D014157:Transcription Factors; D008156:Luciferases; D005982:Glutathione Transferase