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Nucleic Acids Research, 2003, Vol. 31, No. 21 6290-6305
© 2003 Oxford University Press

Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity

Patricia Giraldo1, Antonio Martínez2, Lucía Regales1, Alfonso Lavado1, Angel García-Díaz1, Ángel Alonso3, Ana Busturia2 and Lluís Montoliu*,1

1 Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain, 2 Centro de Biología Molecular ‘Severo Ochoa’ CBM-CSIC/UAM, Campus de Cantoblanco, 28049 Madrid, Spain and 3 Deutsches Krebsforschungszentrum, Themenbereich Infektion und Krebs, Im Neuenheimer Feld, 280, D-69120 Heidelberg, Germany

*To whom correspondence should be addressed. Tel: +34 915854844; Fax: +34 915854506; Email: montoliu{at}cnb.uam.es

Locus control regions (LCRs) are complex high-order chromatin structures harbouring several regulatory elements, including enhancers and boundaries. We have analysed the mouse tyrosinase LCR functions, in vitro, in cell lines and, in vivo, in transgenic mice and flies. The LCR-core (2.1 kb), located at –15 kb and carrying a previously described tissue-specific DNase I hypersensitive site, operates as a transcriptional enhancer that efficiently transactivates heterologous promoters in a cell-specific orientation-independent manner. Furthermore, we have investigated the boundary activity of these sequences in transgenic animals and cells. In mice, the LCR fragment (3.7 kb) rescued a weakly expressed reference construct that displays position effects. In Drosophila, the LCR fragment and its core insulated the expression of a white minigene reporter construct from chromosomal position effects. In cells, sequences located 5' from the LCR-core displayed putative boundary activities. We have obtained genomic sequences surrounding the LCR fragment and found a LINE1 repeated element at 5'. In B16 melanoma and L929 fibroblast mouse cells, this element was found heavily methylated, supporting the existence of putative boundary elements that could prevent the spreading of condensed chromatin from the LINE1 sequences into the LCR fragment, experimentally shown to be in an open chromatin structure.


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