Nucleic Acids Research, 2002, Vol. 30, No. 7 1522-1530
© 2002 Oxford University Press
Requirements for utilization of CREB binding protein by hypersensitive site two of the ß-globin locus control region
University of Wisconsin Medical School, Department of Pharmacology, Molecular and Cellular Pharmacology Program, 383 Medical Science Center, 1300 University Avenue, Madison, WI 53706, USA
Strong transactivation of the ß-globin genes is conferred by the ß-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 A
-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.
* To whom correspondence should be addressed. Tel: +1 608 265 6446; Fax: +1 608 262 1257; Email: ehbresni{at}facstaff.wisc.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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