Non-random asynchronous replication at 22q11.2 favours unequal meiotic crossovers leading to the human 22q11.2 deletion

Background: Analyses of the replication timing at 22q11.2 were prompted by our finding of a statistically significant bias in the origin of the regions flanking the deletion site in patients with 22q11.2 deletions, the proximal region being in the majority of cases of grandmaternal origin. We hypothesised that asynchronous replication may be involved in the formation of the 22q11.2 deletion, the most frequently occurring interstitial deletion in humans, by favouring the mispairing of low-copy repeats.

Methods: Replication timing during S phase at 22q11.2 was investigated by fluorescent in situ hybridisation on interphase nuclei. We report on the detection of non-random asynchronous replication at the human chromosome region 22q11.2, an autosomal locus believed not to contain imprinted genes.

Results: Asynchronous replication at 22q11.2 was observed without exception in all 20 tested individuals; these comprised individuals with structurally normal chromosomes 22 (10 cases), individuals with translocations involving the locus 22q11.2 (eight cases), and patients with a 22q11.2 deletion (two cases). The non-random nature of the asynchronous replication was observed in all individuals for whom the chromosomes 22 were distinguishable. The earlier replicating allele was found to be of paternal origin in all cases where the parental origin of the translocation or deletion was known.

     

Replication timing properties, of the humanHPRT locus on active, inactive and reactivated X chromosomes

X chromosome inactivation is associated with a highly asynchronous pattern of DNA replication at most X-linked loci in females. We studied the human HPRT locus, which is subject to X inactivation and expressed from only the active homolog, with the goal of comparing replication properties between the active and inactive homologs in this region using a fluorescence in situ hybridization approach. We found that in normal female lymphoblasts this locus is replicated in a highly asynchronous manner across a broad, discrete 500–600 kb zone with earliest replication appearing at the gene coding sequence. This general timing profile is maintained in normal male lymphoblasts, as well as in hamster x human hybrid cells containing the active human X chromosome. However, the inactive human X chromosome in the hamster cell background does not appear to function in a fully equivalent manner to the normal inactive X chromosome in female cells. Furthermore, reactivation of the inactive human X chromosome in a hamster x human hybrid system by 5-azacytidine treatment and HAT selection restores early replication at the HPRT gene itself, but does not change the overall domain behavior.     

Replication status as a possible marker for genomic instability in cells originating from genotypes with balanced rearrangements

Most allelic pairs of DNA replicate synchronously during the S phase of the cell cycle. However, some genes frequently replicate asynchronously, i.e. genes on the X chromosome and imprinted genes. Earlier studies demonstrated an asynchronous pattern of replication in some precancerous and invasive squamous carcinoma of the cervix as well as in multiple myeloma. A high rate of asynchronous pattern was found in: (1) lymphocytes of individuals with solid tumors as well as in other malignancies; (2) amniocytes of genotypes with an extra chromosome 13, 18 and 21; (3) lymphocytes of young mothers of a Down syndrome pregnancy. The asynchronic pattern was not locus specific and was found in all loci analyzed. These findings suggested that the mechanism controlling the temporal order of replication could be altered in cells with a genetic predisposition to cancer or aneuploidy. In this study, we found a higher rate of asynchronous pattern in genotypes carrying inversions 2 and 9 and in balanced heritable translocations (p < 0.01) and an even higher rate in cases with a de-novo balanced translocation. The process of tumorigenesis may begin with a change in cell cycle regulation which includes the duplication, replication and segregation of genetic information. However, it remains unknown whether individuals with balanced chromosome rearrangements are at increased risk of developing cancer later in life. This revised version was published online in July 2006 with corrections to the Cover Date.     

The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression.

We have studied replication throughout 325 kb of the telomeric region of a human chromosome (16p13.3) and related the findings to various aspects of chromosome structure and function (DNA sequence organization, nuclease-hypersensitive sites, nuclear matrix attachment sites, patterns of methylation and gene expression). The GC-rich isochore lying adjacent to the telomere, which contains the alpha-globin locus and many widely expressed genes, replicates early in the cell cycle regardless of the pattern of gene expression. In subtelomeric DNA, replication occurs later in the cell cycle and the most telomeric region (20 kb) is late replicating. Juxtaposition of early replicating DNA next to the telomere causes it to replicate later in S-phase. Analysis of the timing of replication in chromosomes with deletions, or in transgenes containing various segments of this telomeric region, suggests that there are no critical origins or zones that initiate replication, rather the pattern of replication appears to be related to the underlying chromatin structure which may restrict or facilitate access to multiple, redundant origins. These results contrast with the pattern of replication at the human beta-globin locus and this may similarly reflect the different chromosomal environments containing these gene clusters.     

Analysis of replication timing at the FRA10B and FRA16B fragile site loci

The molecular basis for the cytogenetic appearance of chromosomal fragile sites is not yet understood. Late replication and further delay of replication at fragile sites expressing alleles has been observed for FRAXA, FRAXE and FRA3B fragile site loci. We analysed the timing of replication at the FRA10B and FRA16B loci to determine whether late replication is a feature which is shared by all fragile sites and, therefore, is a necessary condition for chromosomal fragile site expression. The FRA10B locus was located in a transitional region between early and late zones of replication. Fragile and non-fragile alleles exhibit a similar replication pattern proximal to the repeat, but fragile alleles are delayed relative to non-fragile ones on the distal side. Although fragility at FRA10B appears to be caused by expansion of an AT-rich repeat in the region, replication time near the repeat was similar in fragile and non-fragile alleles. The FRA16B locus was late replicating and appeared to replicate even later on fragile chromosomes. While these observations are compatible with the hypothesis that delayed replication may play a role in fragile site expression, they suggest that replication delay may not need to occur at the expanded repeat region itself in order to be permissive for fragility. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

DNA replication is altered in Immunodeficiency Centromeric instability Facial anomalies (ICF) cells carrying DNMT3B mutations

ICF syndrome is a rare autosomal recessive disorder that is characterized by Immunodeficiency, Centromeric instability, and Facial anomalies. In all, 60% of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In ICF cells, constitutive heterochromatin is hypomethylated and decondensed, metaphase chromosomes undergo rearrangements (mainly involving juxtacentromeric regions), and more than 700 genes are aberrantly expressed. This work shows that DNA replication is also altered in ICF cells: (i) heterochromatic genes replicate earlier in the S-phase; (ii) global replication fork speed is higher; and (iii) S-phase is shorter. These replication defects may result from chromatin changes that modify DNA accessibility to the replication machinery and/or from changes in the expression level of genes involved in DNA replication. This work highlights the interest of using ICF cells as a model to investigate how DNA methylation regulates DNA replication in humans.     

Engineering translocations with delayed replication: evidence for cis control of chromosome replication timing

Certain chromosome rearrangements, found in cancer cells or in cells exposed to ionizing radiation, exhibit a chromosome-wide delay in replication timing (DRT) that is associated with a delay in mitotic chromosome condensation (DMC). We have developed a chromosome engineering strategy that allows the generation of chromosomes with this DRT/DMC phenotype. We found that approximately 10% of inter-chromosomal translocations induced by two distinct mechanisms, site-specific recombination mediated by Cre or non-homologous end joining of DNA double-strand breaks induced by I-Sce1, result in DRT/DMC. Furthermore, on certain balanced translocations only one of the derivative chromosomes displays the phenotype. Finally, we show that the engineered DRT/DMC chromosomes acquire gross chromosomal rearrangements at an increased rate when compared with non-DRT/DMC chromosomes. These results indicate that the DRT/DMC phenotype is not the result of a stochastic process that could occur at any translocation breakpoint or as an epigenetic response to chromosome damage. Instead, our data indicate that the replication timing of certain derivative chromosomes is regulated by a cis-acting mechanism that delays both initiation and completion of DNA synthesis along the entire length of the chromosome. Because chromosomes with DRT/DMC are common in tumor cells and in cells exposed to ionizing radiation, we propose that DRT/DMC represents a common mechanism responsible for the genomic instability found in cancer cells and for the persistent chromosomal instability associated with cells exposed to ionizing radiation.     

Allele-specific late replication and fragility of the most active common fragile site, FRA3B

FRA3B at 3p14.2 is the most active of the common fragile sites in the human genome and is expressed when cells are exposed to the DNA replication inhibitor, aphidicolin. Several lines of evidence suggest that fragile sites are regions of late replication. To elucidate the relationship between the timing of replication across the FRA3B region and its corresponding fragility, we labeled cells with 5-bromo-2'- deoxyuridine (BrdU) and adopted an immunofluorescent procedure to visualize late replicating DNA (BrdU-substituted DNA) in metaphase chromosomes. We also chose 21 markers along the FRA3B region and analyzed the timing of replication using BrdU-labeled DNA from different stages of the cell cycle sorted by flow cytometry. Our results show that there are two distinct alleles that replicate at different stages in the cell cycle and that breaks/gaps preferentially occurred on the chromosome 3 with the late replication allele. These results provide direct evidence that allele-specific late replication is involved in the fragility of the most active common fragile site, FRA3B.      

X inactivation in triploidy and trisomy: the search for autosomal transfactors that choose the active X

Only one X chromosome functions in diploid human cells irrespective of the sex of the individual and the number of X chromosomes. Yet, as we show, more than one X is active in the majority of human triploid cells. Therefore, we suggest that (i) the active X is chosen by repression of its XIST locus, (ii) the repressor is encoded by an autosome and is dosage sensitive, and (iii) the extra dose of this key repressor enables the expression of more than one X in triploid cells. Because autosomal trisomies might help locate the putative dosage sensitive trans-acting factor, we looked for two active X chromosomes in such cells. Previously, we reported that females trisomic for 18 different human autosomes had only one active X and a normal inactive X chromosome. Now we report the effect of triplication of the four autosomes not studied previously; data about these rare trisomies - full or partial - were used to identify autosomal regions relevant to the choice of active X. We find that triplication of the entire chromosomes 5 and 11 and parts of chromosomes 1 and 19 is associated with normal patterns of X inactivation, excluding these as candidate regions. However, females with inherited triplications of 1p21.3-q25.3, 1p31 and 19p13.2-q13.33 were not ascertained. Thus, if a single key dose-sensitive gene induces XIST repression, it could reside in one of these locations. Alternatively, more than one dosage-sensitive autosomal locus is required to form the repressor complex.     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

Loss of alleles at polymorphic loci on chromosome 2 in uveal melanoma

The loss of alleles at loci on specific chromosomes in some malignant tumors, such as retinoblastoma and Wilms' tumor, suggests that recessive mutations are important in their oncogenesis. We postulate that similar mechanisms may be involved in the formation of uveal melanomas. We studied alleles at autosomal loci in uveal melanoma cells and in the constitutional cells from 19 patients who developed the tumors. We observed loss of alleles only at loci on chromosome #2. This suggests that recessive alleles at some chromosome #2 locus may be important in the oncogenesis of uveal melanomas.     

Microcell-mediated chromosome transfer provides evidence that polysomy promotes structural instability in tumor cell chromosomes through asynchronous replication and breakage within late-replicating regions

It was reported earlier that normal chromosome 3 (chr3) transfer into tumor cells of different origin may suppress their ability to grow in SCID mice. Tumorigenicity may be restored by the loss of certain 3p regions. We transferred a normal cell-derived chr3 into cells of a human renal cell carcinoma line and followed the chromosomal changes during in vivo and in vitro growth. In cells cultivated for 6 weeks or more and in the tumors grown in SCID mice, supernumerary chrs3 were always rearranged, accompanied by 3p losses. Unexpectedly, we found that the rearrangements affected not only the transferred exogenous chr3, but also the endogenous chrs3. Other chromosomes that were polysomic in the recipient cells were affected as well, suggesting that polysomy may be associated with structural chromosome instability. The dominant chromosomal aberrations were unbalanced translocations with preferentially pericentromeric breakpoints. The breakpoint distribution on chr3 preferentially affected the pericentromeric 3p11 (8 breaks) and 3p12-13 (5 breaks) regions. The regions 3p14 and 3q26-27 occasionally were involved as well (one break in each case). These four regions were the latest replicating, as shown by BrdU incorporation-based replication banding. Using fluorescence in situ hybridization-based replication timing, we detected asynchronous and incomplete centromere replication in cells with 3 or 4 copies of chr3, but not in cells with 2. We concluded that in tumor cells, asynchronous and incomplete replication of polysomic chromosomal parts is associated with aberrations that have breakpoints within the late-replicating regions. This may explain the increased structural chromosome instability and preferential pericentromeric localization of breakpoints in hyperploid tumors.