Altered Replication Timing Research Articles

Replication timing alterations are associated with mutation acquisition during breast and lung cancer evolution

During each cell cycle, the process of DNA replication timing is tightly regulated to ensure the accurate duplication of the genome. The extent and significance of alterations in this process during malignant transformation have not been extensively explored. Here, we assess the impact of altered replication timing (ART) on cancer evolution by analysing replication-timing sequencing of cancer and normal cell lines and 952 whole-genome sequenced lung and breast tumours. We find that 6%–18% of the cancer genome exhibits ART, with regions with a change from early to late replication displaying an increased mutation rate and distinct mutational signatures. Whereas regions changing from late to early replication contain genes with increased expression and present a preponderance of APOBEC3-mediated mutation clusters and associated driver mutations. We demonstrate that ART occurs relatively early during cancer evolution and that ART may have a stronger correlation with mutation acquisition than alterations in chromatin structure.

Abstract WS2-4: Clinical implications of tumor heterogeneity single cell genomics

Abstract An understanding of how and why somatic mutations accumulate is required to shed light on cancer evolution. DNA replication during each cell cycle is an essential and highly regulated process that ensures the correct duplication of the entire genome. The timing of DNA replication has been indirectly linked to mutation acquisition and genome instability. However, the extent and the importance of altered replication timing (ART) from normal to cancer cells, and whether this process directly influences mutation acquisition during cancer evolution, have not been explored. Here we evaluated the impact of ART by analysing data from 1271 whole-genome sequenced lung (100,000 Genomes Project 1) and breast (560 breast cancer genomes from Nik-Zainal et al. 2) tumours, together with replication-timing sequencing data of multiple cancer and normal cell lines. We find that 6%-18% of the genome is subject to ART in cancer cells. Genomic regions subject to a shift from early to late replication in cancer cells exhibit an increased mutation rate in tumours and are associated with distinct mutational signatures compared to unaltered early replicated regions. In particular, we identify APOBEC3-mediated mutation clusters (omikli events) in unaltered early and late-to-early ART regions, associated with the acquisition of driver mutations. We demonstrate that ART is a relatively early event during the evolution of breast cancer and lung adenocarcinomas. Finally, genes replicated early in cancer but late in normal cells exhibit an up-regulated expression in tumour samples. The incorporation of single-cell DNA sequencing further enables accurate identification of proliferation rates. Taken together, replication timing alterations during malignant transformation are prevalent in cancer and significantly impact the genomic and transcriptomic landscape during tumour evolution. Citation Format: Nnennaya Kanu. Clinical implications of tumor heterogeneity single cell genomics [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr WS2-4.

Abstract 3620: Investigating the role of altered replication timing during tumor evolution in lung and breast cancer

Abstract Introduction: Faithful DNA replication is a key requirement for normal cell division which initiates from replication origins distributed throughout the genome. Not all origins initiate replication at the same time, generating a conserved temporal order of DNA replication (the replication timing program) which is cell and tissue type specific. The extent to which replication timing alters during malignant transformation and the resultant impact on the genomic landscape in cancer remains unclear. Here, we explored the landscape of altered replication timing in lung and breast cancer and the interplay between altered replication timing and genomic cancer evolution. Methods: To characterize replication timing profiles, the Repli-Seq protocol was applied to 4 lung adenocarcinoma, 3 lung squamous cell carcinoma, and 4 breast cancer cell lines, as well as the cell-of-origin of each cancer type. Replication timing was measured as the log2-transformed ratio between early and late replicated reads in 50kb windows. To identify altered replication timing regions the log2-ratio of each cancer cell line was compared to its matched normal. Publicly available WGS and RNA-seq data from patients were used to investigate the relationship between replication timing and genomic alterations. Results: We identified a systematic shift in replication timing from normal to cancer cell lines in 6%-17% of the genome, half of which were classified as late-to-early and half early-to-late replicated. We identified an increase in mutation load in early-to-late replicated regions in comparison to non-altered early replicated regions, whereas the opposite was true for late-to-early replicated regions. We observed a more prevalent shift in the mutational landscape in breast compared to lung cancer, suggesting that most mutations in breast cancer were accumulated after the alteration in replication timing. In addition, we identified known and novel mutational processes active in differently replicated parts of the genome. Furthermore, we observed an enrichment of APOBEC3-associated mutation clusters (omikli events) in early and late-to-early replicated regions. Recurrently gained copy number segments showed an enrichment in not-altered early replicated regions. Also, late-to-early replicated genes exhibited an increase in expression in cancer relative to normal, whereas early-to-late replicated genes exhibited a decrease in expression. Cancer type-specific cancer genes were enriched in late-to-early replicated regions that became up-regulated in cancer, whereas essential genes tended not to change their replication timing during malignant transformation. Conclusion: We identified significant alterations in the replication timing program during malignant transformation which influence the genomic and transcriptomic landscape during tumor evolution. Citation Format: Michelle Dietzen, Haoran Zhai, Olivia Lucas, Sophia Ward, Yanping Guo, Wei Ting-Lu, Oriol Pich, Simone Zaccaria, Charles Swanton, Nicholas McGranahan, Nnennaya Kanu. Investigating the role of altered replication timing during tumor evolution in lung and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3620.

Replication timing organizes epigenome

Epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. [371][1] [1]: /lookup/doi/10.1126/science.aba5545

Abstract 4611: Identifying drivers of mammary tumorigenesis & elucidating the mechanisms of cancer initiation in DNA replication defective Chaos3 mice

Abstract Distinguishing commonly deleted genes as drivers or passengers of human cancers are key to delineating mechanisms of tumor initiation. The “Chaos3” mouse model of spontaneous breast cancer carries a missense mutation in a gene called Mcm4 (minichromosome maintenance 4), causing destabilization of the MCM2-7 replicative helicase, which in turn causes elevated genomic instability. Approximately 80% of female mice homozygous for this mutation in the C3HeB/FeJ strain background develop mammary tumors with an average latency of 12 months. Genomic analysis of these tumors revealed a recurrent subset of genes with copy number alterations such as Arid1a and Nf1. This project aims to: 1) test whether the genes Arid1a and Nf1 act as mammary tumor drivers in the Chaos3 breast cancer model; 2) understand the genome-wide effects of Arid1a loss in mammary tumorigenesis; 3) identifying potential therapeutic targets for Arid1a-deficient mammary tumors; and 4) unravel the mechanism by which destabilization of MCM2-7 replicative helicase leads to cancer onset. To achieve these aims, several experiments such as conditional mutagenesis, CUTandRUN, RNA-seq, and replication timing profiling were conducted to address my aims. Preliminary data indicates that heterozygous loss of Arid1ain Chaos3 mammary tumors produces a distinct transcriptional profile compared to mammary tumors without Arid1a deletion. Additionally, Chaos3 primary cells have markedly altered replication timing patterns in certain regions of the genome that might explain increased susceptibility to mutations. My experiments can validate new candidates like Arid1a as a driver of sporadic breast cancer in humans. In addition, these studies will not only lend insight into how key tumor suppressors are deleted in the Chaos3 model, but also potentially reveal features of genomic regions that are particularly susceptible to genetic or environmental perturbations to DNA replication. The Chaos3 sporadic breast cancer mouse model mimics sporadic human luminal breast cancer and can be used as a tool to better understand the biology and genetics of a common disease that plagues women worldwide. Citation Format: Marquita Winters, John Schimenti. Identifying drivers of mammary tumorigenesis & elucidating the mechanisms of cancer initiation in DNA replication defective Chaos3 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4611.

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.

Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia.

Genome-wide DNA replication timing (RT) profiles reflect the global three-dimensional chromosome architecture of cells. They also provide a comprehensive and unique megabase-scale picture of cellular epigenetic state. Thus, normal differentiation involves reproducible changes in RT, and transformation generally perturbs these, although the potential effects of altered RT on the properties of transformed cells remain largely unknown. A major challenge to interrogating these issues in human acute lymphoid leukemia (ALL) is the low proliferative activity of most of the cells, which may be further reduced in cryopreserved samples and difficult to overcome invitro. In contrast, the ability of many human ALL cell populations to expand when transplanted into highly immunodeficient mice is well documented. To examine the stability of DNA RT profiles of serially passaged xenografts of primary human B- and T-ALL cells, we first devised a method that circumvents the need for bromodeoxyuridine incorporation to distinguish early versus late S-phase cells. Using this and more standard protocols, we found consistently strong retention in xenografts of the original patient-specific RT features. Moreover, in a case in which genomic analyses indicated changing subclonal dynamics in serial passages, the RT profiles tracked concordantly. These results indicate that DNA RT is a relatively stable feature of human ALLs propagated in immunodeficient mice. In addition, they suggest the power of this approach for future interrogation of the origin and consequences of altered DNA RT in ALL.

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.

CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression.

Although several studies have suggested that the functions of heterochromatin regulators may be regulated by post-translational modifications during cell cycle progression, regulation of the histone methyltransferase Suv39H1 is not fully understood. Here, we demonstrate a direct link between Suv39H1 phosphorylation and cell cycle progression. We show that CDK2 phosphorylates Suv39H1 at Ser391 and these phosphorylation levels oscillate during the cell cycle, peaking at S phase and maintained during S-G2-M phase. The CDK2-mediated phosphorylation of Suv39H1 at Ser391 results in preferential dissociation from chromatin. Furthermore, phosphorylation-mediated dissociation of Suv39H1 from chromatin causes an enhanced occupancy of JMJD2A histone demethylase on heterochromatin and alterations in inactive histone marks. Overexpression of phospho-mimic Suv39H1 induces early replication of heterochromatin, suggesting the importance of Suv39H1 phosphorylation in the replication of heterochromatin. Moreover, overexpression of phospho-defective Suv39H1 caused altered replication timing of heterochromatin and increases sensitivity to replication stress. Collectively, our data suggest that phosphorylation-mediated modulation of Suv39H1-chromatin association may be an initial step in heterochromatin replication.

The SKP1-Cul1-F-box and Leucine-rich Repeat Protein 4 (SCF-FbxL4) Ubiquitin Ligase Regulates Lysine Demethylase 4A (KDM4A)/Jumonji Domain-containing 2A (JMJD2A) Protein

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.

Conserved Antagonism between JMJD2A/KDM4A and HP1γ during Cell Cycle Progression

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.

Altered Replication Timing of IGH Alleles in Burkitt's Lymphoma Depends on IGH Breakpoint Position.

Monoallelic expression of immunoglobulin genes is epigenetically regulated and maintained. Asynchronous replication of the IGH alleles has been implicated in allelic exclusion. Usually, the functional IGH allele replicates early in the S-phase of the cell cycle whereas the non-functional allele replicates late. Previously, the intronic enhancer region (Eμ) and the 3′ Cα enhancer region have been designated as putative replication initiation sites. Activity of these replication origins are likely to be involved in regulation of asynchronous replication. By use of interphase and DNA fiber FISH we have performed a detailed analysis of the configuration of the t(8;14) chromosomal translocation in Burkitt's lymphoma patients and cell lines and showed that the breakpoints in all studied cases were perfectly reciprocal without loss of IGH genomic material. An important implication is that in patients and cell lines harboring a breakpoint in a downstream switch region (Sγ or Sα) the Eμ and the 3′ Cα enhancer region are physically separated from each other, whereas in patients and cell lines with the IGH breakpoint located in the JH-region both enhancer regions remain on the der(14) chromosome. We therefore studied the IGH replication timing in Burkitt's lymphoma cell lines harboring different IGH breakpoints by use of BrdU-FISH and interphase FISH on sorted cell cycle fractions. In two cell lines with JH-region breakpoints (Jiyoye and DG-75) the t(8;14) was invariably targeted to the late replicating IGH allele. In contrast, in all three cell lines with switch-region breakpoints (CA-46, Namalwa, BL-65) asynchronous replication of the IGH alleles was lost. As the position of the breakpoint in the MYC locus at 8q24 differed substantially between these cell lines (DG-75 and CA-46: intron 1 of the c-myc gene; Jiyoye, Namalwa, and BL-65: 100 kb to >500 kb centromeric from c-myc) it is unlikely that the position of the 8q24 breakpoint is of crucial influence on IGH replication timing. We speculate that the Eμ and the 3′ Cα enhancer region regulate the IGH replication timing in a cis-acting manner as physical separation of both enhancer regions results in loss of asynchronous replication.

Altered replication timing of the HIRA/Tuple1 locus in the DiGeorge and Velocardiofacial syndromes

DiGeorge and Velocardiofacial syndromes (DGS/VCFS) are endowed by a similar complex phenotype including cardiovascular, craniofacial, and thymic malformations, and are associated with heterozygous deletions of 22q11 chromosomal band. The Typically Deleted Region in the 22q11.21 subband (here called TDR22) is very gene-dense, and the extent of the deletion has been defined precisely in several studies. However, to date there is no evidence for a mechanism of haploinsufficiency that can fully explain the DGS/VCFS phenotype. In this study, we show that the candidate gene HIRA/Tuple1 mapping on the non-deleted TDR22, in DGS/VCFS subjects presents a delayed replication timing. Moreover, we observed an increase in the cell ratio showing the HIRA/Tuple1 locus localised toward the nuclear periphery. It is known that replication timing and nuclear location are generally correlated to the transcription activity of the relative DNA region. We propose that the alteration in the replication/nuclear location pattern of the non-deleted TDR22 indicates an altered gene regulation hence an altered transcritpion in DGS/VCFS.

Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells

Objective Because the effect of granulocyte colony-stimulating factor (G-CSF), which is widely used for allogeneic stem cell transplantation, on DNA function and stability has not yet been unequivocally elucidated, the aim of this study was to determine whether G-CSF leads to epigenetic and/or genetic modifications. Materials and methods Molecular cytogenetic techniques based on fluorescence in situ hybridization technology were used. Results Lymphocytes of G-CSF mobilized donors displayed epigenetic (altered replication timing of alleles) and genetic (aneuploidy) alterations similar to those observed in lymphocytes of cancer patients. Specifically, in the donors' lymphocytes, biallelically expressed genes ( TP53 and AML1) and a repetitive noncoding DNA sequence associated with chromosome segregation ( CEN17) showed loss of synchrony in allelic replication timing (allele-specific replication). Each displayed a highly asynchronous pattern of allelic replication similar to that characterizing monoallelic expressed genes. This non–locus-specific epigenetic phenomenon, which also affects DNA sequences associated with chromosome segregation, was accompanied by aneuploidy. Although the loss of replication synchrony in the lymphocytes of G-CSF mobilized donors was a transient epigenetic modification, aneuploidy remained unchanged. The G-CSF effect also was observed after G-CSF administration in vitro. 5-Azacytidine, a DNA methylation blocking agent, inhibited G-CSF in vitro induction of allele-specific replication. Conclusion G-CSF, probably via changes in DNA methylation capacity, leads to cancer-characteristic DNA modifications in lymphocytes of normal mobilized donors.

Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication.

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodeficiency syndrome and leads to hypomethylation of satellites 2 and 3 in pericentric heterochromatin. This hypomethylation is associated with centromeric decondensation and chromosomal rearrangements, suggesting that these satellite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation status has only been determined with restriction enzymes that cut infrequently at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed for methylation contains an average of 17 CpG sites. The average level of methylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm. Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA strands. Our analysis has also revealed a pattern of methylation specificity, suggesting that some CpGs in the repeat are more prone to methylation than other sites. Variation in satellite 2 methylation among lymphoblasts from different ICF patients has prompted us to determine the frequency of cytogenetic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factors other than DNA methylation appear to play a major role in this phenomenon. Another such factor may be altered replication timing because we have discovered that the hypomethylation of satellite 2 in ICF cultures is associated with advanced replication.

A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere.

Centromere protein A (CENP-A) is an essential centromere-specific histone H3 homologue. Using combined chromatin immunoprecipitation and DNA array analysis, we have defined a 330 kb CENP-A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10). This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through lower-resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP-A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP-A within centromere-specific nucleosomes. The DNA within this domain has a high AT-content comparable to that of alpha-satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid-S phase. Neocentromere formation is accompanied by a replication time lag around but not within the CENP-A binding region, with this lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres.

Association of fragile X syndrome with delayed replication of the FMR1 gene

The fragile X syndrome is commonly associated with mutant alleles of the FMR1 gene that are hypermethylated and have large expansions of CGG repeats. We present data here on the replication timing of FMR1 that confirm predictions of delayed replication of alleles from affected males. The normal FMR1 allele replicates late in S phase, while alleles from affected males replicate later, the major peak of replication occurring in the flow cytometry fraction usually referred to as G2/M. The delayed timing of replication is not the direct result of a single replication fork stalling at the expanded CGG repeat, because delayed replication was observed for regions on both sides of the repeat. The domain of altered replication timing includes sites at least 150 kb 5′ and 34 kb 3′ of the repeat, indicating that genes in addition to FMR1 may be affected.