The complete diploid reference genome of RPE-1 identifies human phased epigenetic landscapes.

Comparative analysis of recent human genome assemblies highlights profound sequence divergence that peaks within polymorphic loci such as centromeres. This raises the question about the adequacy of relying on human reference genomes to accurately analyze sequencing data derived from experimental cell lines. Here, we generated the complete diploid genome assembly for the human retinal epithelial cells (RPE-1), a widely used non-cancer laboratory cell line with a stable karyotype, to use as matched reference for multi-omics sequencing data analysis. Our RPE1v1.0 assembly presents completely phased haplotypes and chromosome-level scaffolds that span centromeres with ultra-high base accuracy (>QV60). We mapped the haplotype-specific genomic variation specific to this cell line including t(Xq;10q), a stable 73.18 Mb duplication of chromosome 10 translocated onto the microdeleted chromosome X telomere t(Xq;10q). Polymorphisms between haplotypes of the same genome reveals genetic and epigenetic variation for all chromosomes, especially at centromeres. The RPE-1 assembly as matched reference genome improves mapping quality of multi-omics reads originating from RPE-1 cells with drastic reduction in alignments mismatches compared to using the most complete human reference to date (CHM13). Leveraging the accuracy achieved using a matched reference, we were able to identify the kinetochore sites at base pair resolution and show unprecedented variation between haplotypes. This work showcases the use of matched reference genomes for multiomics analyses and serves as the foundation for a call to comprehensively assemble experimentally relevant cell lines for widespread application.

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells.

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

Characterization of Chromosomal Instability in Glioblastoma.

Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with high chromosomal instability (CIN). Replication stress is a major cause of CIN that manifests as chromosome rearrangements, fragility, and breaks, including those cytologically expressed within specific chromosome regions named common fragile sites (CFSs). In this work, we characterized the expression of human CFSs in the glioblastoma U-251 MG cell line upon treatment with the inhibitor of DNA polymerase alpha aphidicolin (APH). We observed 52 gaps/breaks located within previously characterized CFSs. We found 17 to be CFSs in GBM cells upon treatment with APH, showing a frequency equal to at least 1% of the total gaps/breaks. We report that two CFSs localized to regions FRA2E (2p13/p12) and FRA2F (2q22) were only found in U-251 MG cells, but not lymphocytes or fibroblasts, after APH treatment. Notably, these glioblastoma-specific CFSs had a relatively high expression compared to the other CFSs with breakage frequency between ∼7 and 9%. Presence of long genes, incomplete replication, and delayed DNA synthesis during mitosis (MiDAS) after APH treatment suggest that an impaired replication process may contribute to this loci-specific fragility in U-251 MG cells. Altogether, our work offers a characterization of common fragile site expression in glioblastoma U-251 MG cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of this incurable cancer.

Genome (in)stability at tandem repeats.

Repeat sequences account for over half of the human genome and represent a significant source of variation that underlies physiological and pathological states. Yet, their study has been hindered due to limitations in short-reads sequencing technology and difficulties in assembly. A important category of repetitive DNA in the human genome is comprised of tandem repeats (TRs), where repetitive units are arranged in a head-to-tail pattern. Compared to other regions of the genome, TRs carry between 10 and 10,000 fold higher mutation rate. There are several mutagenic mechanisms that can give rise to this propensity toward instability, but their precise contribution remains speculative. Given the high degree of homology between these sequences and their arrangement in tandem, once damaged, TRs have an intrinsic propensity to undergo aberrant recombination with non-allelic exchange and generate harmful rearrangements that may undermine the stability of the entire genome. The dynamic mutagenesis at TRs has been found to underlie individual polymorphism associated with neurodegenerative and neuromuscular disorders, as well as complex genetic diseases like cancer and diabetes. Here, we review our current understanding of the surveillance and repair mechanisms operating within these regions, and we describe how alterations in these protective processes can readily trigger mutational signatures found at TRs, ultimately resulting in the pathological correlation between TRs instability and human diseases. Finally, we provide a viewpoint to counter the detrimental effects that TRs pose in light of their selection and conservation, as important drivers of human evolution.

Impaired Replication Timing Promotes Tissue-Specific Expression of Common Fragile Sites.

Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene; interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes' CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.

Analysis of the Association Between TERC and TERT Genetic Variation and Leukocyte Telomere Length and Human Lifespan-A Follow-Up Study.

We investigated the possible influence of TERC and TERT genetic variation and leukocyte telomere length (LTL) on human lifespan. Four polymorphisms of TERT and three polymorphisms of TERC were examined in a sample of elderly subjects (70⁻100 years). After nine years of follow-up, mortality data were collected, and sub-samples of long-lived/not long-lived were defined. TERT VNTR MNS16A L/L genotype and TERT rs2853691 A/G or G/G genotypes were found to be associated with a significantly higher risk to die before the age of 90 years, and with a significantly lower age at death. The association between lifespan and LTL at baseline was analyzed in a subsample of 163 subjects. Age at baseline was inversely associated with LTL (p < 0.0001). Mean LTL was greater in the subjects still living than in those no longer living at follow-up (0.79 T/S ± 0.09 vs 0.63 T/S ± 0.08, p < 0.0001). Comparison of age classes showed that, among the 70⁻79-year-olds, the difference in mean LTL between those still living and those no longer living at follow-up was greater than among the 80⁻90-year-olds. Our data provide evidence that shorter LTL at baseline may predict a shorter lifespan, but the reliability of LTL as a lifespan biomarker seems to be limited to a specific age (70⁻79 years).

RNA polymerase I transcription is modulated by spatial learning in different brain regions.

Long-term memory is accompanied by changes in neuronal morphology and connectivity. These alterations are thought to depend upon new gene expression and protein synthesis over a distributed network of brain structures. Although much evidence supports the idea that the creation of stable, persistent memory traces requires synthesis of new proteins, the role of rRNA transcription and nucleolar activity in learning and memory has hardly been explored. rRNAs needed for protein synthesis result from the activity of two different RNA polymerases, RNA polymerase I and RNA polymerase III, transcribing for 47S RNA and 5S RNA, respectively. In this study, we first investigated the effects of spatial training in the Morris water maze on 47S RNA transcription in the central nervous system, demonstrating bidirectional modulation of its expression over a distributed neural network. We found learning-induced increases in the nucleolar organizer regions in the hippocampus. Finally, we demonstrated that intrahippocampal administrations of CX-5461 (0.6 µg/side), the specific RNA Polymerase I inhibitor, impair the ability of mice to locate the platform in the same task. These results suggest that de novo rRNA transcription is a necessary step for spatial memory consolidation, and that after learning, it occurs in several brain regions with a complex spatiotemporal dynamic. In this study, we demonstrate for the very first time that spatial learning modulates ribosomal RNA transcription in a wide brain circuit, with anatomical specificities in the dynamic of modulation. Together with pharmacological evidences, data presented here support the hypothesis of a necessary role of RNA Pol-I transcription during spatial memory formation. Read the Editorial Highlight for this article on page 673.

Influence of family history of dementia in the development and progression of late-onset Alzheimer's disease.

Family history of dementia (FH) is a recognized risk factor for developing late-onset Alzheimer's disease (AD). We asked whether having FH increases AD risk and influences disease severity (age at onset and cognitive impairment) in 420 AD patients and 109 controls with (FH+) or without (FH-). The relationships of APOE and other AD risk genes with FH were analyzed as well. The proportion of APOE e4 allele carriers was higher among the FH+ than the FH- AD patients (49.6% vs. 38.9%; P = 0.04). The distribution of the risk genotypes of nine AD susceptibility genes previously examined (CHAT, CYP17, CYP19, ESR1, FSHR, P53, P73, P21, PPARG) did not differ between the FH+ and the FH- AD patients, indicating that none contributed significantly to familial clustering of disease. FH was associated with an increased AD risk (odds ratio [OR] 2.71, 95% confidence interval [CI] 1.44-5.09; P = 0.002) independent of carrying the APOE e4 allele (OR 2.61, 95%CI 1.53-4.44; P = 0.0004). Having a first-degree relative or a parent with dementia was significantly associated with AD risk (OR 2.9, 95%CI 1.3-6.4; P = 0.009 and OR 2.7, 95%CI 1.1-6.2; P = 0.02) but having a sibling with dementia was not (OR 1.7, 95%CI 0.2 to 14.7; P = 0.6). Among the FH+ AD patients, having one or both parents affected seemed to raise the risk of earlier onset age (P = 0.02) and greater cognitive impairment (P = 0.02) than having only an affected sibling, whereas having two or more affected relatives did not.

The importance of molecular cytogenetic analysis prior to using cell lines in research: The case of the KG-1a leukemia cell line.

KG-1 and its less differentiated subline KG-1a are leukemia cell lines used in research in a number of laboratories. The karyotypes of the two lines were initially identical. In the following years, further analysis revealed that the cell lines had acquired additional karyotypical abnormalities and differed in the presence of certain typical chromosomal rearrangements. To obtain cytogenetic authentication prior to the use of the two cell lines, we analyzed their karyotype by combining DAPI- and CMA-chromosome bandings and a fluorescence in situ hybridization (FISH)-based approach by using BAC clones useful for the identification of chromosome regions of interest. Sequences of the MYC, PLZF, RARA and BCR genes, that are known to play a critical role in leukemogenesis, and certain BAC clones mapped to five known common fragile sites (CFS) were used for the FISH analysis. A telomeric probe (TTAGGG)n and a set of BAC clones were used to characterize the marker chromosome der(1) that was observed in the cell line KG-1a. The existence of notable differences between the karyotype of the KG-1a cell line previously described, and that described in this study, demonstrate that the use of established cancer cell lines should be preceded by cytogenetic and/or molecular characterization.

Characterization of FRA7B, a human common fragile site mapped at the 7p chromosome terminal region.

Common fragile sites (CFS) are specific regions of the mammalian chromosomes that are particularly prone to gaps and breaks. They are a cause of genome instability, and the location of many CFS correlates with breakpoints of aberrations recurrent in some cancers. The molecular characterization of some CFS has not clarified the causes of their fragility. In this work, by using fluorescence in situ hybridization analysis with BAC and PAC clones, we determined the DNA sequence of the CFS FRA7B. The FRA7B sequence was then analyzed to identify coding sequences and some structural features possibly involved in fragility. FRA7B spans about 12.2 megabases, and is therefore one of the largest CFS analyzed. It maps at the 7p21.3-22.3 chromosome bands, therefore at the interface of G- and R-band regions that are probably difficult to replicate. A 90-kilobase long sequence that presents very high flexibility values was identified at the very beginning of the more fragile CFS region. Three large genes (THSD7A, SDK1, and MAD1L1) and two miRNA genes (MIRN589 and MIRN339) map in the fragile region. The chromosome band 7p22 is a recurrent breakpoint in chromosome abnormalities in different types of neoplasm. FRA7B is the first characterized CFS located in a chromosome terminal region.

Breakages at common fragile sites set boundaries of amplified regions in two leukemia cell lines K562 - Molecular characterization of FRA2H and localization of a new CFS FRA2S.

Genome amplification is often observed in human tumors. The breakage-fusion-bridge (BFB) cycle is the mechanism that often underlies duplicated regions. Some research has indicated common fragile sites (CFS) as possible sites of chromosome breakages at the origin of BFB cycles. Here we searched two human genome regions known as amplification hot spots for any DNA copy number amplifications by analyzing 21 cancer cell lines to investigate the relationship between genomic fragility and amplification. We identified a duplicated region on a chromosomes der(2) present in the karyotype of two analysed leukemia cell lines K562. The two duplicated regions are organized into large palindromes, which suggests that one BFB cycle has occurred. Our findings show that the three breakpoints are localized in the sequence of three CFSs: FRA2H (2q32.1-q32.2), which here has been characterized molecularly; FRA2S (2q22.3-q23.3), a newly localized aphidicolin inducible CFS; and FRA2G (2q24.3-q31).

Transcriptional profiling of genes at the human common fragile site FRA1H in tumor-derived cell lines.

Common fragile sites (CFSs) are chromosome regions that exhibit gaps and breaks when the cells are exposed to replication stress and to some DNA-binding compounds. In cancer cells, the CFSs are frequently involved in recurrent chromosome rearrangements. Furthermore, altered expression of associated genes, known or potential oncogenes, and tumor-suppressor genes has often been observed. Seventeen of the 88 listed CFSs have been analyzed at the molecular level, but the basis of their fragility has not been clarified. In the present work, the nine genes TGFB2, IARS2, MARK1, TAF1A, TP53BP2, ADPRT, including a very large gene ESRRG and two microRNA genes, MIRN194-1 and MIRN215, localized in the fragile site FRA1H, were investigated by polymerase chain reaction (PCR) for homozygous deletions and by real-time PCR for modification or loss of gene expression in a panel of 19 cancer cell lines. The expression level of five (ESRRG, TGFB2, MIRN194-1, MIRN215, and MARK1) of the nine genes studied presented significant modifications in some of the 19 examined tumor-derived cell lines compared to their normal control tissues. Because of their function, these genes could have a role in neoplastic transformation.

Molecular characterization of the human common fragile site FRA1H.

The molecular basis of the fragility of common fragile sites (CFS) and their role in chromosome instability and in altered expression of associated genes in cancer cells have not yet been clarified. In the present work we analyzed the human CFS FRA1H. FRA1H is the first characterized CFS the expression of which is not induced by aphidicolin but instead by DAPI. 5-azaC, 5-azadC, and Ad12 induce a CFS with the same cytogenetic location. By using FISH analysis with BAC clones, we determined that this CFS extends for approximately 10 Mb, and is therefore one of the largest characterized CFSs. FRA1H maps to the chromosome bands 1q41 and 1q42.1 thus spanning an R-band/G-band boundary, a region considered difficult to duplicate. The FRA1H DNA sequence was analyzed to identify coding sequences, the AT content, the type and quantity of the DNA repeats, the CpG islands, the matrix attachment regions, and the number and distribution of high-flexibility regions. A 120 kb long sequence was identified that is very AT-rich (64.6%), has a very large number of flexibility peaks and that may be involved in inducing fragility in the surrounding regions. Among the other genes, two very large genes (USH2A, ESRRG) and two microRNA genes (MIRN194-1, MIRN215) map within the fragile region.

DNA methylation, histone H3 methylation, and histone H4 acetylation in the genome of a crustacean.

In this work, we used antibodies against histone H3 trimethylated at lysine 9 (H3K9m3); against histone H4 acetylated at lysines 5, 8, 12, and 16 (H4ac); and against DNA methylated at 5C cytosine (m5C) to study the presence and distribution of these markers in the genome of the isopod crustacean Asellus aquaticus. The use of these 3 antibodies to immunolabel spermatogonial metaphases yields reproducible patterns on the chromosomes of this crustacean. The X and Y chromosomes present an identical banding pattern with each of the antibodies. The heterochromatic telomeric regions and the centromeric regions are rich in H3K9m3, but depleted in m5C and H4ac. Thus, m5C does not seem to be required to stabilize the silence of these regions in this organism.

Biallelic deletion and loss of expression analysis of genes at FRA2G common fragile site in tumor-derived cell lines.

Common fragile sites (CFS) are regions of chromosome instability that show gaps or breaks when cells are exposed to particular culture condition. Much evidence suggests that CFSs are causally related to cancer as breakpoints in recurrent chromosome mutations and as sites of viral integration. We investigated the FRA2G CFS (2q31) for biallelic deletions and loss of expression in a panel of 19 tumor-derived cell lines. We found that Burkitt lymphoma-derived cell line DAUDI has a biallelic deletion of eight of the nine analyzed genes. Moreover, we observed loss of expression (LOE) of the DHRS9 gene (alias RDHL), one of the deleted genes in the DAUDI cell line, in MOLT-14 and Raji cell lines derived from Burkitt lymphoma and from T-cell acute lymphoblastic leukemia, respectively. DHRS9 is involved in development and differentiation pathways.

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species.

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3 flanking regions, all genes present both the 3 box, a conserved sequence required for 3 processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis.

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site.

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 --> q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.