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Locus Control Region Activity1Department of Biological Sciences, City University of New York, Hunter College, New York, NY 10021
The mouse TCR
/TCR
/Dad1 gene locus bears a locus control region (LCR) that drives high-level, position-independent, thymic transgene expression in chromatin. It achieves this through DNA sequences that enhance transcription and protect transgene expression from integration site-dependent position effects. The former activity maps to a classical enhancer region (E). In contrast, the elements supporting the latter capacity that suppresses position effects are incompletely understood. Such elements likely play important roles in their native locus and may resemble insulator/boundary sequences. Insulators support enhancer blocking and/or chromatin barrier activity. Most vertebrate enhancer-blocking insulators are dependent on the CTCF transcription factor and its cognate DNA binding site. However, studies have also revealed CTCF-independent enhancer blocking and barrier insulator activity in the vertebrate genome. The TCR LCR contains a CTCF-dependent and multiple CTCF-independent enhancer-blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream elements located in the region of the LCR that supports CTCF-independent enhancer-blocking activity in cell culture. Internal deletion of either of these elements significantly impairs LCR activity. These results reveal that the position-effect suppression region of the TCR LCR harbors an array of CTCF-independent, positive-acting gene regulatory elements, some of which share characteristics with barrier-type insulators. These elements may help manage the separate regulatory programs of the TCR and Dad1 genes.
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1 This work was supported by National Science Foundation Career Award MCB-0236964 and National Institutes of Health (NIH) Grant AI-053050 (to B.D.O.). J.G.K. and J.A. were supported by Research Centers in Minority Institutions Award RR-03037 from the National Center for Research Resources of the NIH that also provides support of the infrastructure and instrumentation in the Department of Biological Sciences at Hunter College. F.H. was a Fellow of the Research Initiative for Scientific Enhancement Program (Grant GM-060665) of the NIH and a Ford Foundation Dissertation Fellow.
2 Current address: National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892.
3 Current address: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
4 Address correspondence and reprint requests to Dr. Benjamin D. Ortiz, Department of Biological Sciences, City University of New York, Hunter College, 695 Park Avenue, Room 927N, New York, NY 10021. E-mail address: ortiz{at}genectr.hunter.cuny.edu
5 Abbreviations used in this paper: LCR, locus control region; HS, hypersensitive site.
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  C. Ribeiro de Almeida, H. Heath, S. Krpic, G. M. Dingjan, J. P. van Hamburg, I. Bergen, S. van de Nobelen, F. Sleutels, F. Grosveld, N. Galjart, et al. Critical Role for the Transcription Regulator CCCTC-Binding Factor in the Control of Th2 Cytokine Expression J. Immunol., January 15, 2009; 182(2): 999 - 1010. [Abstract] [Full Text] [PDF]
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