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Molecular and Cellular Biology, April 2001, p. 2629-2640, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2629-2640.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reconstitution of Human beta -Globin Locus Control Region Hypersensitive Sites in the Absence of Chromatin Assembly

K. M. Leach,1 K. Nightingale,2,dagger K. Igarashi,3 P. P. Levings,1 J. D. Engel,4 P. B. Becker,2 and J. Bungert1,*

Department of Biochemistry and Molecular Biology, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida1; Adolf Butenandt Institute, Ludwig Maximillian University, Munich, Germany2; Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima, Japan3; and Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois4

Received 1 September 2000/Returned for modification 26 October 2000/Accepted 24 January 2001

The human beta -globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO4-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.

* Corresponding author. Mailing address: University of Florida, Powell Gene Therapy Center, Department of Biochemistry and Molecular Biology, Gainesville, FL 32610. Phone: (352) 392-0121. Fax: (352) 392-2953. E-mail: jbungert{at}college.med.ufl.edu.

dagger Present address: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.

Molecular and Cellular Biology, April 2001, p. 2629-2640, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2629-2640.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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