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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.

Original publication

DOI

10.1074/jbc.272.16.10975

Type

Journal article

Journal

J Biol Chem

Publication Date

18/04/1997

Volume

272

Pages

10975 - 10980

Keywords

Alleles, Animals, Base Sequence, Blotting, Southern, Cell Line, Crosses, Genetic, DNA, DNA Footprinting, Female, Liver, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Molecular Sequence Data, Polymerase Chain Reaction, Promoter Regions, Genetic, RNA, Long Noncoding, RNA, Untranslated, Restriction Mapping, Sulfuric Acid Esters, Transcription Factors, Ultraviolet Rays, X Chromosome