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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3242-3249
Locus control region activity by 5'HS3 requires a functional
interaction with  -globin gene regulatory elements: expression of
novel / -globin hybrid transgenes
Joel E. Rubin,
Peter Pasceri,
Xiumei Wu,
Philippe Leboulch, and
James Ellis
From the Program in Developmental Biology, and Cancer and Blood
Research Program, Hospital for Sick Children, Toronto, and the
Department of Molecular and Medical Genetics, University of Toronto,
Ontario, Canada; and the Massachusetts Institute of Technology,
Division of Health Sciences & Technology, Cambridge, and Harvard
Medical School and Division of Hematology, Department of Medicine,
Brigham & Women's Hospital, Boston, MA.
The human  -globin locus control region (LCR) contains chromatin
opening and transcriptional enhancement activities that are important
to include in -globin gene therapy vectors. We previously used
single-copy transgenic mice to map chromatin opening activity to the
5'HS3 LCR element. Here, we test novel hybrid globin genes to
identify -globin gene sequences that functionally interact with
5'HS3. First, we show that an 850-base pair (bp) 5'HS3
element activates high-level -globin gene expression in fetal livers of 17 of 17 transgenic mice, including 3 single-copy animals, but fails
to reproducibly activate A -globin transgenes. To identify the
-globin gene sequences required for LCR activity by 5'HS3, we
linked the 815-bp -globin promoter to A-globin coding sequences (BGT34), together with either the -globin intron 2 (BGT35), the -globin 3' enhancer (BGT54), or both intron 2 and the 3'
enhancer (BGT50). Of these transgenes, only BGT50 reproducibly
expresses A-globin RNA (including 7 of 7 single-copy animals,
averaging 71% per copy). Modifications to BGT50 show that LCR activity
is detected after replacing the -globin promoter with the 700-bp A-globin promoter, but is abrogated when an AT-rich region is deleted from -globin intron 2. We conclude that LCR activity by
5'HS3 on globin promoters requires the simultaneous presence of
-globin intron 2 sequences and the 260-bp 3' -globin
enhancer. The BGT50 construct extends the utility of the 5'HS3
element to include erythroid expression of nonadult -globin coding
sequences in transgenic animals and its ability to express antisickling -globin coding sequences at single copy are ideal characteristics for a gene therapy cassette.
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