The timing of XIST replication: dominance of the domain

Contiguous replicons are coordinately replicated and may be organized in temporal-spatial domains with early replication domains containing expressed genes and late ones carrying silent genes. XIST is silent on the active, early replicating X chromosome and expressed from the inactive, late replicating homolog. These circumstances potentially deviate from the aforementioned generalization and make studies of replication timing for XIST of special interest. Although earlier investigations of XIST replication in fibroblasts based on analysis of extracted DNA from cells at different stages of the cell cycle suggested that the silent gene does replicate before the expressed allele, studies using FISH technology produced the opposite results. Because the FISH replication studies could not directly distinguish between the active and inactive X chromosomes in the same cell, we undertook a re-investigation of this question utilizing FISH analysis under conditions that allowed us to make that distinction using cells sorted into different cell cycle stages by flow cytometry. The findings reported here indicate that the silent XIST gene on the active X chromosome does replicate before the expressed allele on the inactive X, supporting the view that the time of a gene's replication is determined by the large, multi-replicon domain in which it is located and not necessarily its expression state.