Differences In Replication Timing Research Articles

The asymmetric distribution of RNA polymerase II and nucleosomes on replicated daughter genomes is caused by differences in replication timing between the lagging and the leading strand

Chromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or whether chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription, and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We found a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNA polymerase II (RNAPII) bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: If transcription and replication are codirectional, the leading strand replicates first; if they are counterdirectional, the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 that impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. We propose that active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state.

Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer

DNA replication timing is known to facilitate the establishment of the epigenome, however, the intimate connection between replication timing and changes to the genome and epigenome in cancer remain largely uncharacterised. Here, we perform Repli-Seq and integrated epigenome analyses and demonstrate that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing. A subset of altered replication timing domains are conserved across cancers from different tissue origins. Notably, late-replicating regions in cancer cells display a loss of DNA methylation, and a switch in heterochromatin features from H3K9me3-marked constitutive to H3K27me3-marked facultative heterochromatin. Finally, analysis of 214 prostate and 35 breast cancer genomes reveal that late-replicating regions are prone to cis and early-replication to trans chromosomal rearrangements. Together, our data suggests that the nature of chromosomal rearrangement in cancer is related to the spatial and temporal positioning and altered epigenetic states of early-replicating compared to late-replicating loci.

Distinctive types of postzygotic single-nucleotide mosaicisms in healthy individuals revealed by genome-wide profiling of multiple organs.

Postzygotic single-nucleotide mosaicisms (pSNMs) have been extensively studied in tumors and are known to play critical roles in tumorigenesis. However, the patterns and origin of pSNMs in normal organs of healthy humans remain largely unknown. Using whole-genome sequencing and ultra-deep amplicon re-sequencing, we identified and validated 164 pSNMs from 27 postmortem organ samples obtained from five healthy donors. The mutant allele fractions ranged from 1.0% to 29.7%. Inter- and intra-organ comparison revealed two distinctive types of pSNMs, with about half originating during early embryogenesis (embryonic pSNMs) and the remaining more likely to result from clonal expansion events that had occurred more recently (clonal expansion pSNMs). Compared to clonal expansion pSNMs, embryonic pSNMs had higher proportion of C>T mutations with elevated mutation rate at CpG sites. We observed differences in replication timing between these two types of pSNMs, with embryonic and clonal expansion pSNMs enriched in early- and late-replicating regions, respectively. An increased number of embryonic pSNMs were located in open chromatin states and topologically associating domains that transcribed embryonically. Our findings provide new insights into the origin and spatial distribution of postzygotic mosaicism during normal human development.

Asynchronous Replication in Lymphocytes from Patients with Inflammatory Bowel Disease and Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD) are associated chronic inflammatory diseases with malignant potential. Loss of replication synchrony during the S-phase of the cell cycle has been shown to be linked to several malignant and premalignant states. This study evaluated temporal differences in replication timing between these diseases. The replication pattern of peripheral blood lymphocytes obtained from patients with PSC and IBD and healthy individuals was analyzed by fluorescence in situ hybridization (FISH) in 2 pairs of alleles, in 15qter and 13qter. Asynchrony was determined by the presence of 1 single and 1 set of double dots in the same cell. Samples from subjects with PSC showed significantly greater temporal differences in replication timing, in contrast to the high level of synchrony observed in samples from healthy individuals (p = 0.045). Samples from IBD patients exhibited a nonsignificant increase in replication asynchrony. We believe that these results reflect impairment in the replication control of structural homologous loci in PSC, and that this phenomenon may be correlated with the inflammation-induced malignant potential of this condition.

Cell line differences in replication timing of human glutamate receptor genes and other large genes associated with neural disease

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (>200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.

DNA replication and transcription programs respond to the same chromatin cues.

DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.

Alterations in replication timing of cancer‐related genes in malignant human breast cancer cells

The replication timing of nine genes commonly involved in cancer was investigated in the MCF10 cell lines for human breast cancer progression. Six of these nine genes are part of a constellation of tumor suppressor genes that play a major role in familial human breast cancer (TP53, ATM, PTEN, CHK2, BRCA1, and BRCA2). Three other genes are involved in a large number of human cancers including breast as either tumor suppressors (RB1 and RAD51) or as an oncogene (cMYC). Five of these nine genes (TP53, RAD51, ATM, PTEN, and cMYC) show significant differences (P < 0.05) in replication timing between MCF10A normal human breast cells and the corresponding malignant MCF10CA1a cells. These differences are specific to the malignant state of the MCF10CA1a cells since there were no significant differences in the replication timing of these genes between normal MCF10A cells and the non-malignant cancer MCF10AT1 cells. Microarray analysis further demonstrated that three of these five genes (TP53, RAD51, and cMYC) showed significant changes in gene expression (≥2-fold) between normal and malignant cells. Our findings demonstrate an alteration in the replication timing of a small subset of cancer-related genes in malignant breast cancer cells. These alterations partially correlate with the major transcriptional changes characteristic of the malignant state in these cells.

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization.

DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

Genome organization, function, and imprinting in Prader-Willi and Angelman syndromes.

The chromosomal region, 15q11-q13, involved in Prader-Willi and Angelman syndromes (PWS and AS) represents a paradigm for understanding the relationships between genome structure, epigenetics, evolution, and function. The PWS/AS region is conserved in organization and function with the homologous mouse chromosome 7C region. However, the primate 4 Mb PWS/AS region is bounded by duplicons derived from an ancestral HERC2 gene and other sequences that may predispose to chromosome rearrangements. Within a 2 Mb imprinted domain, gene function depends on parental origin. Genetic evidence suggests that PWS arises from functional loss of several paternally expressed genes, including those that function as RNAs, and that AS results from loss of maternal UBE3A brain-specific expression. Imprinted expression is coordinately controlled in cis by an imprinting center (IC), a genetic element functional in germline and/or early postzygotic development that regulates the establishment of parental specific allelic differences in replication timing, DNA methylation, and chromatin structure.

Position effect of human telomeric repeats on replication timing.

Telomeres are distinct structures, composed of short, repeated sequences, at the ends of all eukaryotic chromosomes. Telomeres have been shown in yeast to induce late replication in S phase and to silence transcription of neighboring genes. To examine the possibility of similar effects in human chromosomes, we studied cells from a subject with a microdeletion of 130 kb at the end of one copy of chromosome arm 22q, repaired by the addition of telomere repeats. Using fluorescence in situ hybridization of S phase nuclei, a distinct difference was found in the replication timing of the breakpoint region between the intact and truncated copies of chromosome 22. This difference was evident as a shift from middle to late replication time of the breakpoint region adjacent to the repaired telomere. This finding suggests that the human telomere sequence influences activation of adjacent replication origin(s). The difference in replication timing between the two chromosomes was not associated with differences in sensitivity to digestion by DNase I or with methylation of regions immediately adjacent to the breakpoint. Furthermore, both alleles of arylsulfatase A, a gene located at a distance of approximately 54 kb from the breakpoint, were expressed. We conclude that as in yeast, the proximity of telomeric DNA may induce a positional effect that delays the replication of adjacent chromosomal regions in humans.

Temporal differences in replication timing of homologous loci in malignant cells derived from CML and lymphoma patients.

A close association usually exists between replication timing of a given locus and its transcriptional activity: expressed loci replicate early whereas silent ones replicate late. Accordingly, alleles that show concomitant expression replicate synchronously, while those displaying an allele-specific mode of expression show temporal differences in their replication timing, i.e., they replicate asynchronously. We aimed in our study to see whether the cancer phenotype is accompanied by a relaxation in the temporal control of allelic replication. Fluorescence in situ hybridization (FISH) was used to determine the level of synchronization in replication timing of four pairs of homologous loci in samples of malignant cells derived from patients with chronic myeloid leukemia (CML) and lymphoma and in samples from healthy individuals. Four loci, HER2 mapped to 17q11.2-q12, a locus at 21q22, TP53 mapped to 17q13.1, and MYC mapped to 8q24 were studied. In each sample we analyzed two chromosomal regions, either 17q11.2-q12 and 21q22 or 17p13.1 and 8q24. The results showed distinct differences between healthy individuals and CML/lymphoma patients: all samples derived from noncancerous subjects showed high levels of synchrony in replication timing of alleles, whereas those of cancer patients displayed a large temporal difference in replication timing, indicating early and late replicating alleles. Thus, as judged by four unrelated loci, malignancy is associated with changes in the replication pattern of homologous loci.

Asynchronous replication of allelic loci in Down syndrome.

We have used FISH to determine the level of synchronisation in replication timing of four pairs of alleles, unrelated to chromosome 21 (p53, HER2, RB1, and c-myc), in foetal (amniotic fluid) cell samples of Down syndrome and in normal foetuses. All samples derived from the Down syndrome subjects showed large temporal differences in replication timing, in contrast to the high level of synchrony shown in all samples of normal individuals. Thus, as judged by four independent loci which are not associated with chromosome 21, the additional chromosome in the Down syndrome genome induces changes in the replication pattern of an allelic pair: from a synchronous pattern characteristic to concomitantly expressed alleles to an unsynchronised one shown by alleles displaying an allele-specific mode of expression.

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle.

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences.

Parental alleles of an imprinted mouse transgene replicate synchronously.

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting.

The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns.

Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.

Factors affecting the timing and imprinting of replication on a mammalian chromosome

Fluorescence in situ hybridisation has been used to follow replication of the short arm of human chromosome 11 using chromosome anomalies to distinguish the maternally-and paternally-derived homologues. The temporal difference in replication timing within and between chromosomes has been estimated by combining S phase detection with dual colour fluorescence in situ hybridisation. Proximal regions of 11p, including the WT1 gene, tend to replicate earlier on the maternally-derived chromosome than on the paternally-derived homologue. More distal parts of 11p (including the IGF2 gene) have the opposite imprint. The average difference in replication timing between homologous loci in the population of cells is small compared to the differences between loci along a single chromosome. The imprint is not strictly adhered to since many nuclei have hybridisation patterns opposite to the trend within the population. The nature of the imprinting signal has been investigated. Absolute replication time, but not the imprint, was affected by azacytidine, an inhibitor of DNA methylation. The replication imprint was modified by treatments that inhibit histone deacetylation. We suggest that replication imprinting reflects differences in chromatin structure between homologues.

Allele-specific replication timing in imprinted domains: absence of asynchrony at several loci.

Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader-Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing.

Silent substitutions in mammalian genomes and their evolutionary implications.

An analysis of silent substitutions in pairwise comparisons of homologous genes from different mammals has shown that, in spite of individual fluctuations, their frequencies (which are very strongly correlated with the frequency of substitutions per synonymous site calculated according to Li et al. 1985) do not vary, on the average, with the GC levels of silent positions. This holds in the general case, in which silent positions of pairs of homologous genes share the same composition, namely in the human/other primates, human/artiodactyls, and in the mouse/rat pairs, as well as in the special cases in which the composition of silent positions are different, namely in the human/rabbit and the human/rat (or human/mouse) pairs. A slightly lower frequency found for low GC values in the human/bovine and human/pig pairs seems to be due to the specific gene samples used. These results contradict the previously claimed existence of differences in mutation rates and of mutational biases in third codon positions of coding sequences located in different isochores of mammalian genomes. They also imply that the variations in nucleotide precursor pools through the cell cycle and the differences in replication timing, or in repair efficiency, which were reported for different isochores, do not lead, as claimed, to differences in mutation rates, not in mutational biases in mammals. The differences claimed appear to be due to using small gene samples when individual fluctuations from gene to gene are relatively large.

Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

The replication timing of a pair of natural alleles was compared at two alpha-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altA1 allele completes replication at between 20 and 40 min of S phase. The same region of the altA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both altA and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than altA message on Northern (RNA) blots. The temporal windows over which the altA alleles each replicate are very broad in comparison with the levels of mitotic synchrony and altB replication synchrony in a single plasmodium. The allele-specific replication schedule of the altA locus demonstrates that the temporal organization of replicons is not strictly conserved between homologous chromosomes.