The Energetics and Physiological Impact of Cohesin Extrusion.

Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.

Genome Organization Drives Chromosome Fragility.

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability.

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

The 10,000-year biocultural history of fallow deer and its implications for conservation policy.

Over the last 10,000 y, humans have manipulated fallow deer populations with varying outcomes. Persian fallow deer (Dama mesopotamica) are now endangered. European fallow deer (Dama dama) are globally widespread and are simultaneously considered wild, domestic, endangered, invasive and are even the national animal of Barbuda and Antigua. Despite their close association with people, there is no consensus regarding their natural ranges or the timing and circumstances of their human-mediated translocations and extirpations. Our mitochondrial analyses of modern and archaeological specimens revealed two distinct clades of European fallow deer present in Anatolia and the Balkans. Zooarchaeological evidence suggests these regions were their sole glacial refugia. By combining biomolecular analyses with archaeological and textual evidence, we chart the declining distribution of Persian fallow deer and demonstrate that humans repeatedly translocated European fallow deer, sourced from the most geographically distant populations. Deer taken to Neolithic Chios and Rhodes derived not from nearby Anatolia, but from the Balkans. Though fallow deer were translocated throughout the Mediterranean as part of their association with the Greco-Roman goddesses Artemis and Diana, deer taken to Roman Mallorca were not locally available Dama dama, but Dama mesopotamica. Romans also initially introduced fallow deer to Northern Europe but the species became extinct and was reintroduced in the medieval period, this time from Anatolia. European colonial powers then transported deer populations across the globe. The biocultural histories of fallow deer challenge preconceptions about the divisions between wild and domestic species and provide information that should underpin modern management strategies.

Mouse models of human multiple myeloma subgroups.

Multiple myeloma (MM), a tumor of germinal center (GC)-experienced plasma cells, comprises distinct genetic subgroups, such as the t(11;14)/CCND1 and the t(4;14)/MMSET subtype. We have generated genetically defined, subgroup-specific MM models by the GC B cell-specific coactivation of mouse Ccnd1 or MMSET with a constitutively active Ikk2 mutant, mimicking the secondary NF-κB activation frequently seen in human MM. Ccnd1/Ikk2ca and MMSET/Ikk2ca mice developed a pronounced, clonally restricted plasma cell outgrowth with age, accompanied by serum M spikes, bone marrow insufficiency, and bone lesions. The transgenic plasma cells could be propagated in vivo and showed distinct transcriptional profiles, resembling their human MM counterparts. Thus, we show that targeting the expression of genes involved in MM subgroup-specific chromosomal translocations into mouse GC B cells translates into distinct MM-like diseases that recapitulate key features of the human tumors, opening the way to a better understanding of the pathogenesis and therapeutic vulnerabilities of different MM subgroups.

Discovery of fusion circular RNAs in leukemia with KMT2A::AFF1 rearrangements by the new software CircFusion.

Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

The mechanisms of human lymphoid chromosomal translocations and their medical relevance.

The most common human lymphoid chromosomal translocations involve concurrent failures of the recombination activating gene (RAG) complex and Activation-Induced Deaminase (AID). These are two enzymes that are normally expressed for purposes of the two site-specific DNA recombination processes: V(D)J recombination and class switch recombination (CSR). First, though it is rare, a low level of expression of AID can introduce long-lived T:G mismatch lesions at 20-600 bp fragile zones. Second, the V(D)J recombination process can occasionally fail to rejoin coding ends, and this failure may permit an opportunity for Artemis:DNA-dependent kinase catalytic subunit (DNA-PKcs) to convert the T:G mismatch sites at the fragile zones into double-strand breaks. The 20-600 bp fragile zones must be, at least transiently, in a single-stranded DNA (ssDNA) state for the first step to occur, because AID only acts on ssDNA. Here we discuss the key DNA sequence features that lead to AID action at a fragile zone, which are (a) the proximity and density of strings of cytosine nucleotides (C-strings) that cause a B/A-intermediate DNA conformation; (b) overlapping AID hotspots that contain a methyl CpG (WRCG), which AID converts to a long-lived T:G mismatch; and (c) transcription, which, though not essential, favors increased ssDNA in the fragile zone. We also summarize chromosomal features of the focal fragile zones in lymphoid malignancies and discuss the clinical relevance of understanding the translocation mechanisms. Many of the key principles covered here are also relevant to chromosomal translocations in non-lymphoid somatic cells as well.

Next-generation cytogenetics: Comprehensive assessment of 52 hematological malignancy genomes by optical genome mapping.

Somatic structural variants (SVs) are important drivers of cancer development and progression. In a diagnostic set-up, especially for hematological malignancies, the comprehensive analysis of all SVs in a given sample still requires a combination of cytogenetic techniques, including karyotyping, FISH, and CNV microarrays. We hypothesize that the combination of these classical approaches could be replaced by optical genome mapping (OGM). Samples from 52 individuals with a clinical diagnosis of a hematological malignancy, divided into simple (<5 aberrations, n = 36) and complex (≥5 aberrations, n = 16) cases, were processed for OGM, reaching on average: 283-fold genome coverage. OGM called a total of 918 high-confidence SVs per sample, of which, on average, 13 were rare and >100 kb. In addition, on average, 73 CNVs were called per sample, of which six were >5 Mb. For the 36 simple cases, all clinically reported aberrations were detected, including deletions, insertions, inversions, aneuploidies, and translocations. For the 16 complex cases, results were largely concordant between standard-of-care and OGM, but OGM often revealed higher complexity than previously recognized. Detailed technical comparison with standard-of-care tests showed high analytical validity of OGM, resulting in a sensitivity of 100% and a positive predictive value of >80%. Importantly, OGM resulted in a more complete assessment than any previous single test and most likely reported the most accurate underlying genomic architecture (e.g., for complex translocations, chromoanagenesis, and marker chromosomes). In conclusion, the excellent concordance of OGM with diagnostic standard assays demonstrates its potential to replace classical cytogenetic tests as well as to rapidly map novel leukemia drivers.

Molecular tumor board: molecularly adjusted therapy upon identification and functional validation of a novel ALK resistance mutation in a case of lung adenocarcinoma.

We report a case of a long-term surviving patient with EML4/ALK translocated non-small cell adenocarcinoma of the lung in UICC8 stage IVA. During recurrence under continuous crizotinib therapy, a hitherto insufficiently characterized missense mutation in the ALK gene (Arg1181His) was identified through targeted sequencing. The aforementioned EML4/ALK translocation could still be detected in this situation. Employing a 3D reconstruction of the ALK tertiary structure, considering its interaction with various ALK inhibitors at the molecular binding site, our analysis indicated the presence of a mutation associated with crizotinib resistance. To validate the biological relevance of this previously unknown mutation, we carried out an in vitro validation approach in cell culture in addition to the molecular diagnostics accompanied by the molecular tumor board. The tumor scenario was mimicked through retroviral transfection. Our comparative in vitro treatment regimen paired with the clinical trajectory of the patient, corroborated our initial clinical and biochemical suspicions. Our approach demonstrates preclinical, in silico, and clinical evidence of a novel crizotinib resistance mutation in ALK as well as sensitivity toward brigatinib and potentially lorlatinib. In future cases, this procedure represents an important contribution to functional diagnostics in the context of molecular tumor boards.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA.

MAIN CONCLUSION: Agrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes. Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

Plant chromosome engineering - past, present and future.

Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.

Unravelling the mystery of endemic versus translocated populations of the endangered Australian lungfish (Neoceratodus forsteri).

The Australian lungfish is a primitive and endangered representative of the subclass Dipnoi. The distribution of this species is limited to south-east Queensland, with some populations considered endemic and others possibly descending from translocations in the late nineteenth century shortly after European discovery. Attempts to resolve the historical distribution of this species have met with conflicting results based on descriptive genetic studies. Understanding if all populations are endemic or some are the result of, or influenced by, translocation events, has implications for conservation management. In this work, we analysed the genetic variation at three types of markers (mtDNA genomes, 11 STRs and 5196 nuclear SNPs) using the approximate Bayesian computation (ABC) algorithm to compare several demographic models. We postulated different contributions of Mary River and Burnett River gene pools into the Brisbane River and North Pine River populations, related to documented translocation events. We ran the analysis for each marker type separately, and we also estimated the posterior probabilities of the models combining the markers. Nuclear SNPs have the highest power to correctly identify the true model among the simulated datasets (where the model was known), but different marker types typically provided similar answers. The most supported demographic model able to explain the real dataset implies that an endemic gene pool is still present in the Brisbane and North Pine Rivers and coexists with the gene pools derived from past documented translocation events. These results support the view that ABC modelling can be useful to reconstruct complex historical translocation events with contemporary implications, and will inform ongoing conservation efforts for the endangered and iconic Australian lungfish.

Characterizing feral swine movement across the contiguous United States using neural networks and genetic data.

Globalization has led to the frequent movement of species out of their native habitat. Some of these species become highly invasive and capable of profoundly altering invaded ecosystems. Feral swine (Sus scrofa × domesticus) are recognized as being among the most destructive invasive species, with populations established on all continents except Antarctica. Within the United States (US), feral swine are responsible for extensive crop damage, the destruction of native ecosystems, and the spread of disease. Purposeful human-mediated movement of feral swine has contributed to their rapid range expansion over the past 30 years. Patterns of deliberate introduction of feral swine have not been well described as populations may be established or augmented through small, undocumented releases. By leveraging an extensive genomic database of 18,789 samples genotyped at 35,141 single nucleotide polymorphisms (SNPs), we used deep neural networks to identify translocated feral swine across the contiguous US. We classified 20% (3364/16,774) of sampled animals as having been translocated and described general patterns of translocation using measures of centrality in a network analysis. These findings unveil extensive movement of feral swine well beyond their dispersal capabilities, including individuals with predicted origins >1000 km away from their sampling locations. Our study provides insight into the patterns of human-mediated movement of feral swine across the US and from Canada to the northern areas of the US. Further, our study validates the use of neural networks for studying the spread of invasive species.

Genetic prospective of a local invader: the strange pattern of Pontastacus leptodactylus population structure in Greece and Turkey based on microsatellite DNA.

BACKGROUND: The combination of the increasing demand of freshwater crayfish exports, the reduced population sizes due to overfishing, the crayfish plague epidemics and the habitat degradation, have led to unrecorded translocations of Pontastacus leptodactylus in Greek lakes. METHODS AND RESULTS: In the present study, the genetics of five narrow clawed crayfish (P. leptodactylus) populations were studied, namely three translocated populations inhabiting in Northern Greece, one native Greek population from Evros river and one potential progeny source population from Turkey. Nine microsatellite loci previously designed for the specific species were investigated, in order to assess the levels of genetic diversity and further to confirm the origin of these translocated populations some decades after the translocation events. Our results confirmed that the source population for the translocated Greek population is the Turkish lake Egirdir. Further, despite the low values of the number of alleles, heterozygosity, and FST the populations were generally diverse, providing evidence for local adaptation. CONCLUSIONS: The low values of FIS for the translocated populations in combination with the high values of gene flow, possibly indicate the existence of re-introducing events. Apart from the translocated populations, high levels of genetic diversity and heterozygosity were observed in Evros population, suggesting it as a possible unit for future conservation purposes both as a donor population for reintroduction purposes as well as a unique gene pool protection source. To the best of our knowledge this is the first study dealing with the genetic composition of Greek P. leptodactylus populations from Nothern Greece, operating as a first step towards the development of proper management practices for restocking events and monitoring of translocated populations.

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.

Giemsa-negative chromosome bands preferentially recombine in cancer-associated translocations and gene fusions.

Chromosome abnormalities, in particular translocations, and gene fusions are hallmarks of neoplasia. Although both have been recognized as important drivers of cancer for decades, our knowledge of the characterizing features of the cytobands involved in recombinations is poorly understood. The present study, based on a comparative analysis of 10 442 translocation breakpoints and 30 762 gene fusions comprising 13 864 protein-coding genes, is the most comprehensive evaluation of the interactions of cytobands participating in the formation of such rearrangements in cancer. The major conclusion is that although large G-negative, gene-rich bands are most frequently involved, the greatest impact was seen for staining properties. Thus, 60% of the recombinations leading to the formation of both translocations and fusion genes take place between two G-negative bands whereas only about 10% involve two G-positive bands. There is compelling evidence that G-negative bands contain more genes than dark staining bands and it has previously been shown that breakpoints involved in structural chromosome rearrangements and in gene fusions preferentially affect gene-rich bands. The present study not only corroborates these findings but in addition demonstrates that the recombination processes favor the joining of two G-negative cytobands and that this feature may be a stronger factor than gene content. It is reasonable to assume that the formation of translocations and fusion genes in cancer cells, irrespective of whether they have a pathogenetically significant impact or not, may be mediated by some underlying mechanisms that either favor the origin or provide a selective advantage for recombinations of G-negative cytobands.