Role of the region 3' to Xist exon 6 in the counting process of X-chromosome inactivation.
During early embryogenesis of female mammals, one of the two X chromosomes is randomly chosen to be inactivated in each cell, leading to the transcriptional silencing of thousands of genes on this chromosome. This random X-inactivation process also occurs during in vitro differentiation of female embryonic stem (ES) cells. A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', given that at least two copies of Xic must be present per diploid genome in order for inactivation to occur. The counting process ensures that one X chromosome remains active in diploid cells. In the mouse, the essential functions of Xic can be assured by a 450-kb region containing the Xist gene. Xist maps within Xic (refs 7-10) and is necessary in cis for inactivation. The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromosome. In order to characterize functional elements within Xic and the Xist gene, we created a 65-kb cre/loxP deletion extending 3' to Xist exon 6. In undifferentiated ES cells, Xist expression from the deleted X chromosome was markedly reduced. In differentiated XX ES cells containing one deleted X chromosome, the X inactivation process still occurred but was never initiated from the unmutated X chromosome. In differentiated ES cells that were essentially XO, the mutated Xic was capable of initiating X inactivation, even in the absence of another Xic. These results demonstrate a role for the region 3' to Xist exon 6 in the counting process and suggest that counting is mediated by a repressive mechanism which prevents inactivation of a single X chromosome in diploid cells.
Clerc
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P
Génétique Moléculaire Murine, Institut Pasteur, Paris, France. pclerc@pasteur.fr
Avner
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P
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins / Q000235:genetics; D004303:Dosage Compensation, Genetic; D005091:Exons; D005260:Female; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051783:Methyl-CpG-Binding Protein 2; D015778:Minor Histocompatibility Antigens; D015513:Oncogene Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014764:Viral Proteins
D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D015778:Minor Histocompatibility Antigens; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C077885:XIST non-coding RNA; C045073:Cre recombinase; D019426:Integrases