Following chromosome segregation, the nuclear envelope (NE) must be reassembled and holes in the nuclear membrane must be "sealed." During NE assembly, the NE-specific adaptor, Cmp7, recruits/activates ESCRT-III proteins to mediate NE sealing. However, recent evidence suggests the presence of alternative mechanisms. In a screen using the fission yeast, S. japonicus, we recently implicated the ESCRT adaptor, Alx1, and a conserved, but little studied protein, Vid27, in Cmp7-independent NE assembly. Here, we provide direct evidence that Alx1 functions in a Cmp7- and ESCRT-independent NE assembly pathway via positive regulation of Vid27. Consistent with a role in membrane remodeling, Vid27 localizes to sites of postmitotic NE sealing and is essential in S. japonicus. Alx1 and Vid27 form a complex and mutations disrupting their interaction abolish Alx1's enhancement of Vid27 function at the NE. These findings define components of a new Cmp7- and ESCRT-independent NE assembly pathway, advancing our understanding of the mechanisms crucial for maintaining the integrity of the nucleus.

Complex karyotype changes are widespread in cancer genomes. A major gap in cancer genome characterization is the resolution of rearranged chromosomes with chromosome-length continuity. Here, we describe a two-tiered approach to determine the segmental composition of rearranged chromosomes with haplotype resolution. First, we present refLinker, a bioinformatic method for robust determination of chromosomal haplotypes using cancer Hi-C data. By contrast with existing methods, refLinker is insensitive to the presence of large-scale DNA deletions, duplications, and high-level amplification in cancer genomes. Second, we demonstrate a computational strategy to determine the segmental structure of rearranged chromosomes using haplotype-specific Hi-C contacts. We apply these methods to breast cancer genomes and provide direct evidence for long-range transcriptional changes associated with rearrangements of the inactive X chromosome. Together, these results highlight refLinker’s broad utility for studying the functional consequences of chromosomal rearrangements.

Segmental copy-number gains are major contributors to human genetic variation and disease, but how these alterations arise remains incompletely understood. Here, based on the analyses of both experimental evolution and human disease genomes, we describe a general mechanism of segmental copy-number gain from a rearrangement process termed ‘breakage–replication/fusion’. The hallmark genomic feature of breakage–replication/fusion is adjacent parallel breakpoints: two or more rearrangement breakpoints derived from replication of a single ancestral DNA end. We show that adjacent parallel breakpoints are a widespread feature of DNA duplications in human disease genomes and experimental models of chromothripsis. In addition to adjacent parallel breakpoints, breakage–replication/fusion also explains two other patterns of complex rearrangements with unclear provenance: chains of short (≤1 kb) insertions and high-level amplification consisting of inverted segments. Together, these findings revise the mechanistic model for chromothripsis and provide a new conceptual framework for understanding the origin of segmental DNA duplication during genome evolution. This paper introduces breakage–replication/fusion, a genomic rearrangement process underpinning three patterns of copy-number gains found in cancer and other diseases.

Cells entering mitosis with incompletely replicated DNA face catastrophic chromosome segregation failure. During interphase, the replisome-associated E3 ubiquitin ligase TRAIP ubiquitylates barriers in front of the fork to allow replisome progression. In mitosis, TRAIP is reprogrammed from a trans-acting to a cis-acting ligase that can ubiquitylate the replisome itself. This enables the processing of unreplicated DNA by promoting replisome disassembly, fork breakage, and joining of the broken chromosome arms. Here, we describe a mechanism for this reprogramming: The adenosine triphosphatase transcription termination factor 2 (TTF2) is recruited to the replisome, where its noncatalytic N-terminal domain tethers cyclin B-cyclin-dependent kinase 1-phosphorylated TRAIP to the leading strand DNA polymerase ε in a geometry that allows replisome ubiquitylation. Thus, a phosphoregulated architectural switch alters replisome organization in mitosis to safeguard genome integrity before chromosome segregation.

When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.

Genomic characterization has revealed widespread structural complexity in cancer karyotypes, however shotgun sequencing cannot resolve genomic rearrangements with chromosome-length continuity. Here, we describe a two-tiered approach to determine the segmental composition of rearranged chromosomes with haplotype resolution. First, we present refLinker , a new method for robust determination of chromosomal haplotypes using cancer Hi-C data. Second, we use haplotype-specific Hi-C contacts to determine the segmental structure of rearranged chromosomes. By contrast with existing methods for diploid haplotype inference, our approach is robust to the confounding effects of large-scale DNA deletions, duplications, and high-level amplification in cancer sequencing. Using this approach, we examine haplotype-specific expression changes on rearranged homologs and provide direct evidence for long-range transcriptional activation and repression associated with rearrangements of the inactive X chromosome (Xi). Together, these results reveal the significant transcriptional consequences of somatic Xi rearrangements, highlighting refLinker 's broad utility for studying the functional consequences of chromosomal rearrangements.

DNA rearrangements are thought to arise from two classes of processes. The first class involves DNA breakage and fusion ("cut-and-paste") without net DNA gain or loss. The second class involves aberrant DNA replication ("copy-and-paste") and can produce either net DNA gain or loss. We previously demonstrated that the partitioning of chromosomes into aberrant structures of the nucleus, micronuclei or chromosome bridges, can generate cut-and-paste rearrangements by chromosome fragmentation and ligation. Surprisingly, in the progeny clones of single cells that have undergone chromosome bridge breakage, we identified large segmental duplications and short sequence insertions that are commonly attributed to copy-and-paste processes. Here, we demonstrate that both large duplications and short insertions are inherent outcomes of the replication and fusion of unligated DNA ends, a process we term breakage-replication/fusion (B-R/F). We propose that B-R/F provides a unifying explanation for complex rearrangement patterns including chromothripsis and chromoanasynthesis and enables rapid DNA amplification after chromosome fragmentation.

Nuclear envelope (NE) assembly defects cause chromosome fragmentation, cancer, and aging. However, major questions about the mechanism of NE assembly and its relationship to nuclear pathology are unre-solved. In particular, how cells efficiently assemble the NE starting from vastly different, cell type-specific endoplasmic reticulum (ER) morphologies is unclear. Here, we identify a NE assembly mechanism, "mem-brane infiltration,"that defines one end of a continuum with another NE assembly mechanism, "lateral sheet expansion,"in human cells. Membrane infiltration involves the recruitment of ER tubules or small sheets to the chromatin surface by mitotic actin filaments. Lateral sheet expansion involves actin-independent envelopment of peripheral chromatin by large ER sheets that then extend over chromatin within the spindle. We propose a "tubule-sheet continuum"model that explains the efficient NE assembly from any starting ER morphology, the cell type-specific patterns of nuclear pore complex (NPC) assembly, and the obligatory NPC assembly defect of micronuclei.

Supplementary Table S4 shows synthetic lethal analysis using RIGER-E Log Fold Change 2nd Best and STARS algorithm.

This document contains the text for the supplementary data.

Genome evolution can happen gradually or via bursts of rearrangements. Chromoplexy is an example of a process driving rapid genome evolution. This mutational signature is detected in ~18% of human cancers (PCAWG Consortium, 2020) and is frequently observed in prostate adenocarcinoma, lymphoid malignancies, and thyroid adenocarcinoma. Chromoplexy is inferred to happen as one catastrophic event that generates copy-neutral chains of translocations involving multiple chromosomes (Baca et al., 2013). Existing studies of chromoplexy monitor the outcome of massive cancer genome reorganization, thus early molecular events leading to catastrophic chromosome rearrangements remain elusive. In this work, we aimed to recapitulate molecular mechanisms underlying chromoplexy. For this, we set out to establish a cell line model and use fluorescence-based reporter systems to enrich for and allow isolation of cells containing signatures of chromoplexy. We additionally address whether colocalization of multiple double-strand breaks, for example in transcription hubs or abnormal nuclear structures, might stimulate chained inter- and intra- chromosomal translocations typical for chromoplexy. If successful, this work will provide a mechanistic understanding of an important mutational process driving rapid genome evolution in cancer, congenital disease, and potentially organismal evolution. Citation Format: Nataliia Serbyn, Myrthe M. Smit, Vimathi S. Gummalla, Gregory J. Brunette, David S. Pellman. Unravelling the mechanistic basis of chromoplexy, a mutational process driving early cancer genome evolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6105.

Supplementary Table S3 shows enrichment analysis from our screen analyzed using RIGER-E Log Fold Change 2nd Best algorithm.

Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure(1-3) and the evolutionary trajectories(4) of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.

Supplementary Table S5 shows RNA sequencing data and gene set enrichment analysis of NCI-H82 cells Dox-On pRB cells grown in the presence or absence of DOX and then treated with DMSO or AZD2811.

Supplementary Table S1 shows complete list of genes targeted by our custom sgRNA library and sgRNA sequences.

Supplementary Table S6 shows mutational status of RB1 and TP53 and amplification status of MYC in cell line xenograft and PDX SCLC mouse models.

Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance 1 – 3 . However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer 4 , 5 , as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.