KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Dynamic interplay between human alpha-satellite DNA structure and centromere functions.

Maintenance of genome stability relies on functional centromeres for correct chromosome segregation and faithful inheritance of the genetic information. The human centromere is the primary constriction within mitotic chromosomes made up of repetitive alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. Notably, centromeric DNA is faithfully inherited as intact haplotypes from the parents to the offspring without intervening recombination, yet, outside of meiosis, centromeres are akin to common fragile sites (CFSs), manifesting crossing-overs and ongoing sequence instability. Consequences of DNA changes within the centromere are just starting to emerge, with unclear effects on intra- and inter-generational inheritance driven by centromere's essential role in kinetochore assembly. Here, we review evidence of meiotic selection operating to mitigate centromere drive, as well as recent reports on centromere damage, recombination and repair during the mitotic cell division. We propose an antagonistic pleiotropy interpretation to reconcile centromere DNA instability as both driver of aneuploidy that underlies degenerative diseases, while also potentially necessary for the maintenance of homogenized HORs for centromere function. We attempt to provide a framework for this conceptual leap taking into consideration the structural interface of centromere-kinetochore interaction and present case scenarios for its malfunctioning. Finally, we offer an integrated working model to connect DNA instability, chromatin, and structural changes with functional consequences on chromosome integrity.

Emerging roles of DNA repair factors in the stability of centromeres.

Satellite DNA sequences are an integral part of centromeres, regions critical for faithful segregation of chromosomes during cell division. Because of their complex repetitive structure, satellite DNA may act as a barrier to DNA replication and other DNA based transactions ultimately resulting in chromosome breakage. Over the past two decades, several DNA repair proteins have been shown to bind and function at centromeres. While the importance of these repair factors is highlighted by various structural and numerical chromosome aberrations resulting from their inactivation, their roles in helping to maintain genome stability by solving the intrinsic difficulties of satellite DNA replication or promoting their repair are just starting to emerge. In this review, we summarize the current knowledge on the role of DNA repair and DNA damage response proteins in maintaining the structure and function of centromeres in different contexts. We also report the recent connection between the roles of specific DNA repair factors at these genomic loci with age-related increase of chromosomal instability under physiological and pathological conditions.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

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.

Decoding human cancer with whole genome sequencing: a review of PCAWG Project studies published in February 2020.

Cancer is underlined by genetic changes. In an unprecedented international effort, the Pan-Cancer Analysis of Whole Genomes (PCAWG) of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) sequenced the tumors of over two thousand five hundred patients across 38 different cancer types, as well as the corresponding healthy tissue, with the aim of identifying genome-wide mutations exclusively found in cancer and uncovering new genetic changes that drive tumor formation. What set this project apart from earlier efforts is the use of whole genome sequencing (WGS) that enabled to explore alterations beyond the coding DNA, into cancer's non-coding genome. WGS of the entire cohort allowed to tease apart driving mutations that initiate and support carcinogenesis from passenger mutations that do not play an overt role in the disease. At least one causative mutation was found in 95% of all cancers, with many tumors showing an average of 5 driver mutations. The PCAWG Project also assessed the transcriptional output altered in cancer and rebuilt the evolutionary history of each tumor showing that initial driver mutations can occur years if not decades prior to a diagnosis. Here, I provide a concise review of the Pan-Cancer Project papers published on February 2020, along with key computational tools and the digital framework generated as part of the project. This represents an historic effort by hundreds of international collaborators, which provides a comprehensive understanding of cancer genetics, with publicly available data and resources representing a treasure trove of information to advance cancer research for years to come.

HELLS and CDCA7 comprise a bipartite nucleosome remodeling complex defective in ICF syndrome.

Mutations in CDCA7, the SNF2 family protein HELLS (LSH), or the DNA methyltransferase DNMT3b cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome. While it has been speculated that DNA methylation defects cause this disease, little is known about the molecular function of CDCA7 and its functional relationship to HELLS and DNMT3b. Systematic analysis of how the cell cycle, H3K9 methylation, and the mitotic kinase Aurora B affect proteomic profiles of chromatin in Xenopus egg extracts revealed that HELLS and CDCA7 form a stoichiometric complex on chromatin, in a manner sensitive to Aurora B. Although HELLS alone fails to remodel nucleosomes, we demonstrate that the HELLS-CDCA7 complex possesses nucleosome remodeling activity. Furthermore, CDCA7 is essential for loading HELLS onto chromatin, and CDCA7 harboring patient ICF mutations fails to recruit the complex to chromatin. Together, our study identifies a unique bipartite nucleosome remodeling complex where the functional remodeling activity is split between two proteins and thus delineates the defective pathway in ICF syndrome.

Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T.

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

γH2AX as a marker of DNA double strand breaks and genomic instability in human population studies.

DNA double strand breaks (DSB) are the gravest form of DNA damage in eukaryotic cells. Failure to detect DSB and activate appropriate DNA damage responses can cause genomic instability, leading to tumorigenesis and possibly accelerated aging. Phosphorylated histone H2AX (γH2AX) is used as a biomarker of cellular response to DSB and its potential for monitoring DNA damage and repair in human populations has been explored in this review. A systematic search was conducted in PubMed for articles, in English, on human studies reporting γH2AX as a biomarker of either DNA repair or DNA damage. A total of 68 publications were identified. Thirty-four studies (50.0%) evaluated the effect of medical procedures or treatments on γH2AX levels; 20 (29.4%) monitored γH2AX in specific pathological conditions with a case/control or case/case design; 5 studies (7.4%) evaluated the effect of environmental genotoxic exposures, and 9 (13.2%) were descriptive studies on cancer and aging. Peripheral blood lymphocytes (44.6%) or biopsies/tissue specimens (24.3%) were the most commonly used samples. γH2AX was scored by optical microscopy as immunostained foci (78%), or by flow cytometry (16%). Critical features affecting the reliability of the assay, including protocols heterogeneity, specimen, cell cycle, kinetics, study design, and statistical analysis, are hereby discussed. Because of its sensitivity, efficiency and mechanistic relevance, the γH2AX assay has great potential as a DNA damage biomarker; however, the technical and epidemiological heterogeneity highlighted in this review infer a necessity for experimental standardization of the assay.

Visualization of the three-dimensional structure of the human centromere in mitotic chromosomes by superresolution microscopy.

The human centromere comprises large arrays of repetitive α-satellite DNA at the primary constriction of mitotic chromosomes. In addition, centromeres are epigenetically specified by the centromere-specific histone H3 variant CENP-A that supports kinetochore assembly to enable chromosome segregation. Because CENP-A is bound to only a fraction of the α-satellite elements within the megabase-sized centromere DNA, correlating the three-dimensional (3D) organization of α-satellite DNA and CENP-A remains elusive. To visualize centromere organization within a single chromatid, we used a combination of the centromere chromosome orientation fluorescence in situ hybridization (Cen-CO-FISH) technique together with structured illumination microscopy. Cen-CO-FISH allows the differential labeling of the sister chromatids without the denaturation step used in conventional FISH that may affect DNA structure. Our data indicate that α-satellite DNA is arranged in a ring-like organization within prometaphase chromosomes, in the presence or absence of spindle's microtubules. Using expansion microscopy, we found that CENP-A organization within mitotic chromosomes follows a rounded pattern similar to that of α-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Collectively, our data provide a 3D reconstruction of α-satellite DNA along with CENP-A clusters that outlines the overall architecture of the mitotic centromere.

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.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

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.

Centromeres under Pressure: Evolutionary Innovation in Conflict with Conserved Function.

Centromeres are essential genetic elements that enable spindle microtubule attachment for chromosome segregation during mitosis and meiosis. While this function is preserved across species, centromeres display an array of dynamic features, including: (1) rapidly evolving DNA; (2) wide evolutionary diversity in size, shape and organization; (3) evidence of mutational processes to generate homogenized repetitive arrays that characterize centromeres in several species; (4) tolerance to changes in position, as in the case of neocentromeres; and (5) intrinsic fragility derived by sequence composition and secondary DNA structures. Centromere drive underlies rapid centromere DNA evolution due to the "selfish" pursuit to bias meiotic transmission and promote the propagation of stronger centromeres. Yet, the origins of other dynamic features of centromeres remain unclear. Here, we review our current understanding of centromere evolution and plasticity. We also detail the mutagenic processes proposed to shape the divergent genetic nature of centromeres. Changes to centromeres are not simply evolutionary relics, but ongoing shifts that on one side promote centromere flexibility, but on the other can undermine centromere integrity and function with potential pathological implications such as genome instability.

Epigenetics as an Evolutionary Tool for Centromere Flexibility.

Centromeres are the complex structures responsible for the proper segregation of chromosomes during cell division. Structural or functional alterations of the centromere cause aneuploidies and other chromosomal aberrations that can induce cell death with consequences on health and survival of the organism as a whole. Because of their essential function in the cell, centromeres have evolved high flexibility and mechanisms of tolerance to preserve their function following stress, whether it is originating from within or outside the cell. Here, we review the main epigenetic mechanisms of centromeres' adaptability to preserve their functional stability, with particular reference to neocentromeres and holocentromeres. The centromere position can shift in response to altered chromosome structures, but how and why neocentromeres appear in a given chromosome region are still open questions. Models of neocentromere formation developed during the last few years will be hereby discussed. Moreover, we will discuss the evolutionary significance of diffuse centromeres (holocentromeres) in organisms such as nematodes. Despite the differences in DNA sequences, protein composition and centromere size, all of these diverse centromere structures promote efficient chromosome segregation, balancing genome stability and adaptability, and ensuring faithful genome inheritance at each cellular generation.

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.

Interplay of the nuclear envelope with chromatin in physiology and pathology.

The nuclear envelope compartmentalizes chromatin in eukaryotic cells. The main nuclear envelope components are lamins that associate with a panoply of factors, including the LEM domain proteins. The nuclear envelope of mammalian cells opens up during cell division. It is reassembled and associated with chromatin at the end of mitosis when telomeres tether to the nuclear periphery. Lamins, LEM domain proteins, and DNA binding factors, as BAF, contribute to the reorganization of chromatin. In this context, an emerging role is that of the ESCRT complex, a machinery operating in multiple membrane assembly pathways, including nuclear envelope reformation. Research in this area is unraveling how, mechanistically, ESCRTs link to nuclear envelope associated factors as LEM domain proteins. Importantly, ESCRTs work also during interphase for repairing nuclear envelope ruptures. Altogether the advances in this field are giving new clues for the interpretation of diseases implicating nuclear envelope fragility, as laminopathies and cancer. ABBREVIATIONS: na, not analyzed; ko, knockout; kd, knockdown; NE, nuclear envelope; LEM, LAP2-emerin-MAN1 (LEM)-domain containing proteins; LINC, linker of nucleoskeleton and cytoskeleton complexes; Cyt, cytoplasm; Chr, chromatin; MB, midbody; End, endosomes; Tel, telomeres; INM, inner nuclear membrane; NP, nucleoplasm; NPC, Nuclear Pore Complex; ER, Endoplasmic Reticulum; SPB, spindle pole body.

A gust of WNT: analysis of the canonical WNT pathway.

The Wnt pathway is a signal-transduction cascade that mediates communication between cells; the Wnt pathway is involved in key steps during embryological development and in the maintenance of adult tissue homeostasis. Mutational dysregulation of Wnt cascade components has been observed in diverse human pathological conditions and in oncogenic transformations. For these reasons, the Wnt signalling pathway has acquired growing interest in scientific and medical research over recent years. This review outlines the biochemical and functional features of the Wnt cascade with particular emphasis on a detailed functional analysis of all key players. In this instance, the regulations of the pathway have also been covered, emphasizing novelty in this regard. Furthermore, past and present studies on Wnt have been included, as well as a prediction of scientific progress, which may be made in this rapidly evolving field, in the near future; the review also embraces considerations on how further understanding of the Wnt pathway will provide important insight into managing human diseases.