A central role for a single c-Myb binding site in a thymic locus control region.
Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.
Ess
K C
KC
Department of Pediatrics, Children's Hospital Medical Center, University of Cincinnati, Ohio 45229, USA.
Whitaker
T L
TL
Cost
G J
GJ
Witte
D P
DP
Hutton
J J
JJ
Aronow
B J
BJ
D000243:Adenosine Deaminase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013601:T-Lymphocytes; D013950:Thymus Gland / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism*
D002843:Chromatin; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D000243:Adenosine Deaminase