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 chromos omes is randomly chosen to be inactivated in each cell(1), leading to the transcriptional silencing of thousands of genes on this chromosome . This random X-inactivation process also occurs during in vitro diffe rentiation of female embryonic stem (ES) cells(2-4). A locus on the X chromosome, the X inactivation centre (Xic) is initially 'counted', gi ven that at least two copies of Xic must be present per diploid genome in order for inactivation to occur(2). The counting process ensures t hat one X chromosome remains active in diploid cells, In the mouse, th e essential functions of Xic can be assured by a 450-kb region(5,6) co ntaining the Xist gene. Xist maps within Xic (refs 7-10) and is necess ary in cis for inactivation(11,12), The Xist transcript is a 15-kb RNA which is confined within the nucleus and coats the inactive X chromos ome(13). In order to characterize functional elements within Xic and t he Xist gene, we created a 65-kb cre/loxP deletion extending 3' to Xis t exon 6, In undifferentiated ES cells, Xist expression from the delet ed X chromosome was markedly reduced. in differentiated XX ES cells co ntaining one deleted X chromosome, the X inactivation process still oc curred but was never initiated from the unmutated X chromosome, In dif ferentiated ES cells that were essentially XO, the mutated Xic was cap able 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 repres sive mechanism which prevents inactivation of a single X chromosome in diploid cells.