Nucleic Acids Research, 1992, Vol. 20, No. 10 2533-2540
© 1992
MOLECULAR BIOLOGY |
Two upstream elements activate transcription of a major histocompatibility complex class I gene in vitro
Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, MD 20892, USA
* To whom correspondence should be addressed
Received January 14, 1992. Revised April 1, 1992. Accepted April 1, 1992.
Expression of major histocompatibility complex (MHC) class I genes exhibits unique tissue and developmental specificity. In an effort to study molecular mechanisms of MHC class I gene regulation, an in vitro transcription system has been established. In B cell nuclear extracts a template DNA containing the mouse H-2Ld promoter sequence accurately directed RNA polymerase II-dependent transcription of a G-free cassette. A conserved class I regulatory complex previously shown to moderately enhance promoter activity in vivo enhanced transcription in vitro by 2–3 fold. Much of this enhancement was accounted for by a 40 bp fragment within the complex, which was capable of activating a basal H-2Ld promoter in either orientation. Farther downstream, another element called site B was identified, which independently activated MHC class I transcription in vitro by 2–4 fold. Site B bound a specific nuclear factor(s) through an NF-1 binding site but not through a neighboring CCAAT site. The functional significance of site B in vivo was demonstrated in transfection experiments in which site B enhanced MHC class I promoter activity to a degree comparable to that seen in vitro. With the identification of the two upstream activators, MHC class I genes may serve as a model to study roles of sequence-specific DNA-binding proteins in transcription in vitro.
Present address: +Department of Molecular Biology, Lewis Thomas Laboratories, Princeton University, Princeton, NJ 08544
Present address: 
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75240, USA
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