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Molecular and Cellular Biology, May 2001, p. 2969-2980, Vol. 21, No. 9
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.9.2969-2980.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Sequences Flanking Hypersensitive Sites of the beta -Globin Locus Control Region Are Required for Synergistic Enhancement

Joseph M. Molete,1 Hanna Petrykowska,1 Eric E. Bouhassira,3 Yong-Qing Feng,3 Webb Miller,2 and Ross C. Hardison1,*

Department of Biochemistry and Molecular Biology1 and Department of Computer Science and Engineering,2 The Pennsylvania State University, University Park, Pennsylvania, and Department of Medicine, Division of Hematology, Albert Einstein College of Medicine, Bronx, New York3

Received 19 October 2000/Returned for modification 19 December 2000/Accepted 9 February 2001

The major distal regulatory sequence for the beta -globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.

* Corresponding author. Mailing address: 206 Althouse Lab, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-0113. Fax: (814) 863-7024. E-mail: rch8{at}psu.edu.

Molecular and Cellular Biology, May 2001, p. 2969-2980, Vol. 21, No. 9
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.9.2969-2980.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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