Analysis of replication timing properties of human X-chromosomal loci by fluorescence in situ hybridization.

Abstract

We have used fluorescence in situ hybridization on interphase nuclei of normal female cells to compare the replication timing patterns of genes on the human X chromosome that are known to escape X inactivation with those that are inactivated. By this procedure it was possible not only to determine the relative time of replication of the earlierreplicating allele for different loci but also to estimate the degree of asynchrony of replication of the two alleles for each individual locus. Loci such as HPRT and FRAXA, which are normally inactivated, displayed a high degree of replication asynchrony, whereas loci that are not inactivated (ZFX and RPS4X) were found to replicate very synchronously. Interestingly, examination of XIST, which is expressed only from the inactive X chromosome, by this procedure revealed that it also replicated asynchronously, with the expressed copy apparently replicating first. Therefore, by examining different loci from the X chromosome it was determined that there is a strict correlation between the expression and relative time of replication of individual genes.One of the distinctive hallmarks of X chromosomes in female mammalian cells is their asynchronous, or allocyclic, replication patterns during S phase, which was first noted >30 years ago (1-4). It was quickly recognized that the laterreplicating chromosome was also the one that had undergone the process of X inactivation, which involved transcriptional silencing of the genes on this chromosome and was postulated to be a mechanism of gene dosage compensation between males and females (5,6). This inactivated chromosome was also known to have an altered chromatin structure, associated with the formation ofa heterochromatic Barr body in interphase cells (7). In recent years it has become evident that in human female cells not all the individual genes on the "inactive" chromosome actually undergo the inactivation phenomenon (reviewed in ref. 8). Thus, it is important to test the correlations between gene expression, replication timing, and chromatin structure at the subchromosomal level.In the past, analysis of replication patterns of individual X-linked genes in female cells has been inhibited by the intrinsic asynchronous timing of the two homologs. However, recently a technique was developed by Selig et al. (9) that is not subject to this limitation. In this procedure unsynchronized cells are subjected to in situ hybridization with nonradioactive probes, with subsequent detection by fluorescence methods. By analyzing interphase nuclei one can rapidly determine the replication state of specific chromosomal regions; prior to replication in S phase single hybridization dots are detected, but after replication doublets are seen. Therefore, it is possible to determine the relative order of replication of different alleles and different loci by comparing the singlet/doublet ratios (9, 10).We have used this technique to examine the replication properties of regions encompassing several X-linked genes that undergo the normal transc...

SV40 T antigen interacts with Nbs1 to disrupt DNA replication control

“…FISH was performed as described (Heng et al 1992;Heng and Tsui 1993;Boggs and Chinault 1997). Human BAC clones that were used as probes for DNA sequences at the B2-Lamin, HSP70, and ␤-globin loci were obtained through the Children's Hospital Oakland Research Institute (CHORI).…”

Asynchronous replication of allelic loci in Down syndrome

“…Using this method it was clearly shown that a pair of alleles which are known to be expressed concomitantly replicate synchronously, [10][11][12][13] whilst alleles subjected to some mechanism leading to alelle-specific expression, such as imprinting, 11,[14][15][16] X chromosome inactivation, 12 or some other allelic inactivation, 13,17 replicate asynchronously.…”

S-adenosylhomocysteine treatment of adult female fibroblasts alters X-chromosome inactivation and improves in vitro embryo development after somatic cell nuclear transfer

“…Interestingly, several of the genes, including XIST, showed a notable, albeit non-significant, increase in the level of expression in treated samples. HPRT is transcribed only from the active X-chromosome, but RPS4X and ZFX, both of which had expression levels that were unchanged by treatment, have functional Y-homologs, and are transcribed from both the active and inactive X-chromosome (Boggs & Chinault 1994, Carrel et al 1996, Brown & Chow 2003. It is likely that gene loci escaping X-inactivation such as RPS4X and ZFX are not methylated on either of the X-chromosomes and hence were not affected by DNA demethylation with SAH, whereas the transcript levels of other X-linked genes were slightly increased by SAH treatment.…”