JBC Ideal method for primary cell transfection

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Volume 272, Number 16, Issue of April 18, 1997 pp. 10975-10980
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

In Vivo Ultraviolet and Dimethyl Sulfate Footprinting of the 5' Region of the Expressed and Silent Xist Alleles

(Received for publication, December 6, 1996, and in revised form, February 14, 1997)

Jun-ichiro Komura Dagger , Steven A. Sheardown § , Neil Brockdorff § , Judith Singer-Sam Dagger and Arthur D. Riggs Dagger

From the Dagger  Biology Department, Beckman Research Institute of the City of Hope, Duarte, California 91010 and the § Section of Comparative Biology, Medical Research Council Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 ONN, United Kingdom

The Xist (X inactive specific transcript) gene plays an essential role in X chromosome inactivation. To elucidate the mechanisms controlling Xist expression and X inactivation, we examined in vivo DNA-protein interactions in the Xist promoter region in a female mouse cell line (BMSL2), which has distinguishable Xist alleles. In vivo footprinting was accomplished by treatment of cells with dimethyl sulfate or ultraviolet light, followed by ligation-mediated polymerase chain reaction of purified DNA. The expressed allele on the inactive X chromosome and the silent allele on the active X chromosome were separated by the use of a restriction fragment length polymorphism prior to ligation-mediated polymerase chain reaction. The chromatin structure of the Xist promoter was found to be consistent with the activity state of the Xist gene. The silent allele (on the active X chromosome) showed no footprints, while the expressed allele (on the inactive X chromosome) showed footprints at a consensus sequence for a CCAAT box, two weak Sp1 sites, and a weak TATA box.


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