In vivo ultraviolet and dimethyl sulfate footprinting of the 5' region of the expressed and silent Xist alleles.
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.
Komura
J i
Ji
Biology Department, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Sheardown
S A
SA
Brockdorff
N
N
Singer-Sam
J
J
Riggs
A D
AD
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003433:Crosses, Genetic; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification / Q000528:radiation effects; D018983:DNA Footprinting / Q000379:methods; D005260:Female; D008099:Liver / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000528:radiation effects; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D015183:Restriction Mapping; D013463:Sulfuric Acid Esters; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics*; D014466:Ultraviolet Rays; D014960:X Chromosome
D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013463:Sulfuric Acid Esters; D014157:Transcription Factors; C077885:XIST non-coding RNA; D004247:DNA; C007482:dimethyl sulfate