title: Requirements for utilization of CREB binding protein by hypersensitive site two of the beta-globin locus control region.
abstract: Strong transactivation of the beta-globin genes is conferred by the beta-globin locus control region (LCR), which consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4). HS2 has a powerful enhancer activity dependent upon tandem binding sites for the erythroid cell- and megakaryocyte-specific transcription factor NF-E2. An important co-activator-mediating transactivation by HS2 is the histone acetyltransferase (HAT) CREB binding protein (CBP). We showed previously that recruitment of a GAL4-CBP fusion protein to HS2 largely bypassed the requirement of the NF-E2 sites for transactivation. To determine whether GAL4-CBP recruitment is sufficient for transactivation, we assessed the importance of cis-elements within HS2. Docking of GAL4-CBP upstream of an Agamma-globin promoter lacking HS2 only weakly activated the promoter, indicating that HS2 components are required for GAL4-CBP-mediated transactivation. Sequences upstream and downstream of the NF-E2 sites were required for maximal GAL4-CBP-mediated transactivation, and HAT catalytic activity of GAL4-CBP was critical. No single factor-binding site was required for GAL4-CBP-mediated transactivation. However, deletion of two sites, a CACC site and an E-box, abolished transactivation in transient and stable transfection assays. These results suggest that NF-E2 recruits CBP as a critical step in transactivation, but additional components of HS2 are required to achieve maximal enhancer activity.
authors.affiliation: University of Wisconsin Medical School, Department of Pharmacology, Molecular and Cellular Pharmacology Program, 383 Medical Science Center, 1300 University Avenue, Madison, WI 53706, USA.
authors.forename: Kirby D
authors.initials: KD
authors.lastname: Johnson
authors.forename: Jason E
authors.initials: JE
authors.lastname: Norton
authors.forename: Emery H
authors.initials: EH
authors.lastname: Bresnick
mesh_terms: D000123:Acetyltransferases / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D050882:CREB-Binding Protein; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050981:Erythroid-Specific DNA-Binding Factors; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D051548:Histone Acetyltransferases; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D005914:Globins; D000123:Acetyltransferases; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D051548:Histone Acetyltransferases