|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Mammalian X chromosome inactivation is a chromosome-wide phenomenon that leads to transcriptional silencing of one of the two X chromosomes in female somatic cells. The inactivated X chromosome shares striking properties, including asynchronous DNA replication, XIST RNA localization through the length of the X chromosome, sex chromatin body formation at nuclear periphery, and hypoacetylation of histone H4. Relationships among these properties and their relative importance, however, remain unknown. In the present study, we have tried to isolate trans-acting factors involved in heterochromatinization of the inactive human X chromosome.
A human monochromosome hybrid, CF150 has one or more inactive human X chromosomes (Xi) as its only human element. In CF150 cells, the introduced Xi behaved abnormally ; (1) Xi did not form sex chromatin body at nucleus peripherally, (2) Xi was moderately acetylated as other mouse chromosomes, and (3) Xi was not coated with XIST RNA.The same Xi transferred f
rom CF150 into HeLa cells behaved normally as the typical inactive X, suggesting full manifestation of these traits requires a certain human specific factor (s) that is depleted in CF150. A genetic complementation assay has been performed to determine which human chromosome bears the related genes, by microcell fusion.
When Xi was introduced from CF150 into mouse embryonal carcinoma (EC) cell lines, the reversal of inactivation occurred, but XIST expressed persistently. Despite of its active chromatin state, the reactivated human X chromosome was coated with XIST RNA.Cell differentiation of these monochromosome EC hybrids leaded the dissociation of XIST RNA from the human X chromosome. When the same reactivated X chromosome was transferred from monochromosome EC hybrids into HeLa cells, the reactivated X chromosome was coated with XIST RNA.Together, these data suggest that two different factors (an undifferentiated and a somatic cell types, respectively) for Xist RNA localization exist in mouse cells. Less