H4 acetylation, XIST RNA and replication timing are coincident and define x;autosome boundaries in two abnormal X chromosomes

The inactive X (Xi) differs from its active homologue (Xa) in a number ofways, including increased methylation of CpG islands, replication late in Sphase, underacetylation of histone H4 and association with XIST RNA. Globalchanges in DNA methylation occur relatively late in development, but theother properties all change during or shortly after the establishment of Xiand may play a role in the mechanism by which an inactive chromatinconformation spreads across most of the chromosome. In the present report,we use two human X;autosome translocation chromosomes to study thespreading of inactive X chromatin across X;autosome boundaries. In one ofthese chromosomes, t(X;6), Xp distal to p11.2 is replaced by 6p21.1-6pterand, in the other, ins(X;16), a small fragment derived from 16p13 isinserted into the distal third of Xq. In lymphoid cells from patientscarrying these translocations in an unbalanced form, Xi was shown by HUMARAassay to be derived exclusively [t(X:6)] or predominantly [ins (X;16)] fromthe derived X chromosome. We used a combination of immunolabelling andRNA/DNA fluorescence in situ hybridization to define the distribution ofXIST RNA, deacetylated H4 and late-replicating DNA across the two derived Xchromosomes in inactive form. Within the limits of the cytogenetictechniques employed, the results show complete coincidence of these threeparameters, with all three being excluded from the autosomal component ofthe derived X chromosome.