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 of w ays, including increased methylation of CPG islands, replication late in S phase, underacetylation of histone H4 and association with XIST RNA. Global changes in DNA methylation occur relatively late in development, but the o ther properties all change during or shortly after the establishment of Xi and may play a role in the mechanism by which an inactive chromatin conform ation spreads across most of the chromosome. In the present report, we use two human X;autosome translocation chromosomes to study the spreading of in active X chromatin across X;autosome boundaries. In one of these chromosome s, t(X;6), Xp distal to p11.2 is replaced by 6p21.1-6pter and, in the other , ins(X;16), a small fragment derived from 16p13 is inserted into the dista l third of Xq, In lymphoid cells from patients carrying these translocation s in an unbalanced form, Xi was shown by HUMARA assay to be derived exclusi vely [t(X:6)] or predominantly [ins (X;16)] from the derived X chromosome. We used a combination of immunolabelling and RNA/DNA fluorescence in situ h ybridization to define the distribution of XIST RNA, deacetylated H4 and la te-replicating DNA across the two derived X chromosomes in inactive form. W ithin the limits of the cytogenetic techniques employed, the results show c omplete coincidence of these three parameters, with all three being exclude d from the autosomal component of the derived X chromosome.