DNA-REPLICATION ANALYSIS OF FMRI, XIST, AND FACTOR 8C LOCI BY FISH SHOWS NONTRANSCRIBED X-LINKED GENES REPLICATE LATE

The relationship between the transcriptional state of a locus and the time when it replicates during DNA synthesis is increasingly apparent. Active autosomal genes tend to replicate early, whereas inactive ones are more permissive and frequently replicate later. Although the inac tive X chromosome replicates later than its active homologue, little i s known about the replication of X-linked genes, We have used FISH to examine the replication of loci on the active X chromosome that are no t transcribed, either because the tissue analyzed was not the expressi ng tissue (F8C), because the locus is silent on all active X chromosom es (XIST), or because it has been mutated by expansion and methylation of a CpG island (FMR1). In this assay, an unreplicated locus is chara cterized by a single hybridization signal, and a replicated locus is c haracterized by a doublet hybridization signal. The percentage of doub lets is used as a measure of relative time of replication in S phase. The validity of this approach has been established elsewhere, since re sults compare favorably with those obtained using traditional methods for studying DNA replication. Our results show that the FMR1 gene repl icates relatively later in fragile X (fraX) males with the full mutati on than in normal males, irrespective of the probe used. The F8C locus is late replicating in both normal and fraX males and replicates at n early the same time on active and inactive X in females. The XIST locu s replicates late in all the males studied and asynchronously in femal e cells. From the late replication of the locus on the active X in mal es, we deduce that the locus on the active X is the later replicating locus in female cells. We conclude that (1) the expansion of the FMR1 locus leads to late replication, (2) silence of the XIST gene in males is associated with late replication of the locus, and (3) this assay will be useful for further studies of the relationship between transcr iption and replication.