Rearrangements In Human Cells Research Articles

Mechanisms of insertions at a DNA double-strand break

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells

Clastogen exposure can result in chromosomal rearrangements, including large deletions and inversions that are associated with cancer development. To examine such rearrangements in human cells, here we developed a reporter assay based on endogenous genes on chromosome 12. Using the RNA-guided nuclease Cas9, we induced two DNA double-strand breaks, one each in the GAPDH and CD4 genes, that caused a deletion rearrangement leading to CD4 expression from the GAPDH promoter. We observed that this GAPDH-CD4 deletion rearrangement activates CD4+ cells that can be readily detected by flow cytometry. Similarly, double-strand breaks in the LPCAT3 and CD4 genes induced an LPCAT3-CD4 inversion rearrangement resulting in CD4 expression. Studying the GAPDH-CD4 deletion rearrangement in multiple cell lines, we found that the canonical non-homologous end joining (C-NHEJ) factor XLF promotes these rearrangements. Junction analysis uncovered that the relative contribution of C-NHEJ appears lower in U2OS than in HEK293 and A549 cells. Furthermore, an ATM kinase inhibitor increased C-NHEJ-mediated rearrangements only in U2OS cells. We also found that an XLF residue that is critical for an interaction with the C-NHEJ factor X-ray repair cross-complementing 4 (XRCC4), and XRCC4 itself are each important for promoting both this deletion rearrangement and end joining without insertion/deletion mutations. In summary, a reporter assay based on endogenous genes on chromosome 12 reveals that XLF-dependent C-NHEJ promotes deletion rearrangements in human cells and that cell type-specific differences in the contribution of C-NHEJ and ATM kinase inhibition influence these rearrangements.

Nuclear myosin/actin-motored contact between homologous chromosomes is initiated by ATM kinase and homology-directed repair proteins at double-strand DNA breaks to suppress chromosome rearrangements.

We provide evidence for a mechanism of DNA repair that requires nuclear myosin/actin-dependent contact between homologous chromosomes to prevent formation of chromosomal rearrangement in human cells. We recently showed that DNA double strand breaks (DSBs) induced by γ-rays or endonucleases cause ATM-dependent contact formation between homologous chromosomes at damaged sites of transcriptionally active chromatin in G0/G1-phase cells. Here, we report that the mechanism of contact generation between homologous chromosomes also requires homology-directed repair proteins, including BRCA1, RAD51 and RAD52, and nuclear myosin/actin-motors. Moreover, inhibition of ATM kinase or deficiency in nuclear actin polymerization causes carcinogenic RET/PTC chromosome rearrangements after DSBs induction in human cells. These data suggest that DSBs in transcriptionally active euchromatin in G0/G1-phase cells are repaired through a mechanism that requires contact formation between homologous chromosomes and that this mechanism is mediated by HDR proteins and nuclear myosin/actin motors.

PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA

Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. Here we develop a zinc-finger nuclease translocation reporter and screen for factors that modulate rearrangements in human cells. We identify UBC9 and RAD50 as suppressors and 53BP1, DDB1 and poly(ADP)ribose polymerase 3 (PARP3) as promoters of chromosomal rearrangements across human cell types. We focus on PARP3 as it is dispensable for murine viability and has druggable catalytic activity. We find that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage, which suppresses repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3−/− cells leads to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways.

98-03-03 ヒト細胞の染色体組換えに関与するDNA損傷の性状(平成10年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

98-03-03 ヒト細胞の染色体組換えに関与するDNA損傷の性状(平成10年度プロジェクト研究報告(保健学部・保健学研究科),研究助成金による成果紹介)

Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells

It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However, the actual distance between these fusion partners has never been determined. The phenomenon of fluorescence resonance energy transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-sensitized emission (SE) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements.

Suitability of Animal Models for Studying Radiation-Induced Thyroid Cancer in Humans: Evidence from Nuclear Architecture

Rat and mouse have been widely used to estimate the radiation risk and tumorigenic effects of radiation with extrapolating the findings to humans. RET/PTC is a characteristic genetic alteration frequently found in radiation-induced thyroid cancer in human populations. Recently, nuclear architecture and spatial proximity between recombinogenic genes have been implicated as important factors in the generation of RET/PTC and other chromosomal rearrangements in human cells. However, it is unknown whether the nuclear architecture in rodent thyroid cells is similar to that of human thyroid cells. The aim of this study was to test whether the proximity effects that are observed between loci involved in RET/PTC rearrangements in humans are conserved across different species. Using 3D fixation, fluorescence in situ hybridization, and confocal microscopy, we compared the distance between genes involved in RET/PTC rearrangement in normal thyroid cells from humans, mice, and rats. While in humans, RET, NCOA4, and H4 are all located on the same chromosome (10q), in rodents these genes are located on separate chromosomes. In mouse, RET is located on chromosome 6F1, NCOA4 on 14B, and H4 on 10B5.3. In rat, RET is on chromosome 4q42, NCOA4 on 16p16, and H4 (TST1) on 9q36. We further observed that in human thyroid cells, mean distance between genes involved in two most common types of RET/PTC, that is, RET and NCOA4 (partners of RET/PTC3) and RET and H4 (partners of RET/PTC1), was 1.08±0.04 and 1.24±0.05 μm, respectively. In mouse thyroid cells, these distances were 3.21±0.1 and 3.43±0.1 μm, and in rat cells the values were 3.37±0.1 and 3.87±0.1 μm (p<0.001). Moreover, we found that in contrast to human thyroid cells, in rodent cells these genes were randomly positioned with respect to each other. The differences in nuclear architecture and spatial positioning of genes involved in RET/PTC rearrangements between human and rodent thyroid cells raise a concern about suitability of animal models for assessing RET/PTC-driven thyroid carcinogenesis in humans.

Influence of Nuclear Geometry on the Formation of Genetic Rearrangements in Human Cells

Interphase chromosomes are divided into discrete domains, with limited overlapping and movement. We explored the role of nuclear topology in the formation of chromosome aberrations by irradiating normal human fibroblasts with high-energy heavy ions from different directions. Cells with elliptical nuclei were grown in an aligned manner onto micrometer grooved culturing substrates to have a predetermined orientation with respect to the accelerated iron ions. Particles were directed either perpendicular to the cell layer or along the major or minor axis of the nucleus. Analysis of chromosome aberrations by mFISH showed that, at the same radiation dose, the yield of chromosomal damage and its complexity are largely modified by the irradiation geometry. The results demonstrate that the architecture of the cell nucleus determines the formation of chromosomal rearrangements.

DNA breaks at fragile sites generate oncogenic RET/PTC rearrangements in human thyroid cells

Common fragile sites are regions of the genome that are prone to DNA breakage and frequently coincide with the location of genes involved in carcinogenic chromosomal translocations. In this study, we examined the direct involvement of fragile sites in the formation of RET/PTC rearrangements, which are commonly found in papillary thyroid carcinomas, and are all a result of a translocation event involving the RET gene. We demonstrated that fragile site‐inducing chemicals, including aphidicolin, can induce the formation of RET/PTC rearrangements in thyroid cells. We further studied fragile site‐induced DNA breakage within the genes (RET, CCDC6, and NCOA4) participating in the two major types of RET/PTC rearrangements, RET/PTC1 and RET/PTC3, through cytogenetic, molecular biology, and sequence analysis methods. Using FISH, we found the rate of DNA breakage at these genes to be similar to known fragile site‐induction levels of the common fragile sites containing these genes, FRA10C and FRA10G. We mapped the location of induced breakpoints using LM‐PCR and found them to be located near breakpoints observed in patients. Factors contributing to the formation of DNA breaks have been investigated using sequence analysis. These results provide the first direct evidence for the role of chromosomal fragile sites in the generation of cancer‐specific rearrangements in human cells. (Supported by the NCI, CA113863).

DNA breaks at fragile sites generate oncogenic RET/PTC rearrangements in human thyroid cells

Human chromosomal fragile sites are regions of the genome that are prone to DNA breakage, and are classified as common or rare, depending on their frequency in the population. Common fragile sites frequently coincide with the location of genes involved in carcinogenic chromosomal translocations, suggesting their role in cancer formation. However, there has been no direct evidence linking breakage at fragile sites to the formation of a cancer-specific translocation. Here, we studied the involvement of fragile sites in the formation of RET/PTC rearrangements, which are frequently found in papillary thyroid carcinoma (PTC). These rearrangements are commonly associated with radiation exposure; however most of the tumors found in adults are not linked to radiation. In this study, we provide structural and biochemical evidence that the RET, CCDC6, and NCOA4 genes participating in two major types of RET/PTC rearrangements, are located in common fragile sites FRA10C and FRA10G, and undergo DNA breakage after exposure to fragile site-inducing chemicals. Moreover, exposure of human thyroid cells to these chemicals results in the formation of cancer-specific RET/PTC rearrangements. These results provide the direct evidence for the involvement of chromosomal fragile sites in the generation of cancer-specific rearrangements in human cells.

Enhanced Sensitivity of the RET Proto-Oncogene to Ionizing Radiation In vitro

Exposure to ionizing radiation is a well-known risk factor for a number of human cancers, including leukemia and thyroid cancer. It has been known for a long time that exposure of cells to radiation results in extensive DNA damage; however, a small number of studies have tried to explain the mechanisms of radiation-induced carcinogenesis. The high prevalence of RET/PTC rearrangements in patients who have received external radiation, and the evidence of in vitro induction of RET rearrangements in human cells, suggest an enhanced sensitivity of the RET genomic region to damage by ionizing radiation. To assess whether RET is indeed more sensitive to radiations than other genomic regions, we used a COMET assay coupled with fluorescence in situ hybridization, which allows the measurement of DNA fragmentation in defined genomic regions of single cells. We compared the initial DNA damage of the genomic regions of RET, CXCL12/SDF1, ABL, MYC, PLA2G2A, p53, and JAK2 induced by ionizing radiation in both a lymphoblastoid and a fetal thyroid cell line. In both cell lines, RET fragmentation was significantly higher than in other genomic regions. Moreover, a differential distribution of signals within the COMET was associated with a higher percentage of RET fragments in the tail. RET was more susceptible to fragmentation in the thyroid-derived cells than in lymphoblasts. This enhanced susceptibility of RET to ionizing radiation suggests the possibility of using it as a radiation exposure marker.

Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells.

Rearrangements involving the RET gene are common in radiation-associated papillary thyroid cancer (PTC). The RET/PTC1 type of rearrangement is an inversion of chromosome 10 mediated by illegitimate recombination between the RET and the H4 genes, which are 30 megabases apart. Here we ask whether despite the great linear distance between them, RET and H4 recombination might be promoted by their proximity in the nucleus. We used two-color fluorescence in situ hybridization and three-dimensional microscopy to map the positions of the RET and H4 loci within interphase nuclei. At least one pair of RET and H4 was juxtaposed in 35% of normal human thyroid cells and in 21% of peripheral blood lymphocytes, but only in 6% of normal mammary epithelial cells. Spatial contiguity of RET and H4 may provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double-strand break in each gene at the same site in the nucleus.

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Correlation between repair patch ligation and nucleosome rearrangement in human cells treated with bleomycin, UV radiation or methyl methanesulfonate.

We have examined ligation and nucleosome rearrangement of repair patches in chromatin of human fibroblasts damaged with bleomycin (BLM), UV radiation and methyl methanesulfonate (MMS) to follow completion of excision repair involving different combinations of repair enzymes. Conditions were used that allowed analysis of the correlation between these two events over a large range (i.e. from 5% to greater than 99% ligated). Cells exposed to BLM were reversibly permeabilized with L-alpha-lysophosphatidylcholine and pulse-labeled with either [3H]dTTP or [3H]dThd to label selectively cells that 'reseal their membranes' at different rates. A striking difference is observed in the rates of ligation of these nascent repair patches, in that those labeled with [3H]dTTP are ligated much slower (25-50% unligated after 24 h) than those labeled with [3H]dThd (less than 5% unligated after 6 h). The nuclease sensitivity of [3H]dTTP-labeled patches also decreases more slowly, indicating that the rate of nucleosome rearrangement decreases compared to that of repair patches labeled with [3H]dThd. The rates of repair patch ligation and loss of nuclease sensitivity were also modulated in intact cells exposed to UV radiation or MMS by treatment with aphidicolin and/or hydroxyurea. A plot of relative nuclease sensitivity versus fraction of ligated repair patches yields an overall linear correlation of greater than 0.8 in each case, indicating that these two features of nascent repair patches are 'moderately coupled' events. These results support the idea that ligation of repair patches is a prerequisite for nucleosome rearrangement following three different modes of excision repair.

Nucleosome rearrangement in human cells following short patch repair of DNA damaged by bleomycin

We have examined the structure of newly repaired regions of chromatin in intact and permeabilized human cells following exposure to bleomycin (BLM). The average repair patch size (in permeabilized cells) was six to nine bases, following doses of 1-25 micrograms/mL BLM, and greater than 80% of the total repair synthesis was resistant to aphidicolin. In both intact and permeabilized cells, nascent repair patches were initially very sensitive to staphylococcal nuclease, analogous to repair induced by "long patch" agents, and are nearly absent from isolated nucleosome core DNA. Unlike long patch repair, however, the loss of nuclease sensitivity during subsequent chase periods was very slow in intact cells, or in permeabilized cells treated with a low dose of BLM (1 microgram/mL), and was abolished by treatment with hydroxyurea (HU) or aphidicolin (APC). The rate of repair patch ligation did not correlate with this slow rate of chromatin rearrangement since greater than 95% of the patches were ligated within 6 h after incorporation (even in the presence of HU or APC). In permeabilized cells, repair patches induced by either 5 or 25 micrograms/mL BLM, where significant levels of strand breaks occur in compact regions of chromatin, lost the enhanced nuclease sensitivity at a rate similar to that observed following long patch repair. This rapid rate of rearrangement was not affected by APC. These results indicate that short patch repair in linker regions of nucleosomes, and/or "open" regions of chromatin, involves much less nucleosome rearrangement than long patch repair or short patch repair in condensed chromatin domains.

Inducible Gene Expression by DNA Rearrangements in Human Cells

A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency.

Inducible gene expression by DNA rearrangements in human cells.

A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency.

Nuclease sensitivity of repair-incorporated nucleotides in chromatin and nucleosome rearrangement in human cells damaged by methyl methanesulfonate and methylnitrosourea.

We have examined both the initial nuclease sensitivity and subsequent nucleosome rearrangement of newly repaired regions of chromatin in human diploid fibroblasts treated with methyl methanesulfonate (MMS) and methylnitrosourea (MNU). We initially examined the effect of these two alkylating agents on DNA replicative synthesis. The results indicate that immediately following damage by MMS or MNU, at a concentration of 2 mM, the level of replicative synthesis is 20-25% of the level in untreated cells. In the MMS-treated cells, this suppression of replicative synthesis is short lived and by 15 h after damage the level of replicative synthesis is approximately 3-fold greater than that in untreated cells. This 'latent stimulation' of replicative synthesis was not observed in the cells treated with 2 mM MNU, although the level of replicative synthesis in these cells did approach the level of untreated cells at later times. When these contributions were corrected for, it was found that the nucleotides incorporated by repair synthesis are initially (i.e., immediately following repair synthesis) both staphylococcal nuclease and DNase I sensitive, and are underrepresented in isolated nucleosome core DNA. Using methods previously described by us, we show that the relative nuclease sensitivity of these regions is quantitatively similar to that of newly repaired DNA following damage by u.v. radiation. Furthermore, the relative nuclease sensitivity of newly repaired DNA is initially high regardless of the time after damage that repair occurs (at least for 13 h after damage). This feature is also similar to u.v. induced repair synthesis. Finally, pulse-chase experiments demonstrated that following repair synthesis induced by MMS or MNU rearrangements of chromatin structure take place, and both the rate and extent of these rearrangements are similar to that observed for cells treated with u.v. radiation or bulky chemical carcinogens. Thus, our results indicate that the excision repair induced by these two small alkylating agents is associated with the same overall chromatin structural features as the excision repair of DNA damage induced by u.v. radiation and 'u.v.-mimetic' chemicals.