Biology subjects

Pellman, D.

Publications and source records attributed to Pellman, D..

11 recordsLinked to original sources

bioRxiv2026

The TIRR–53BP1 axis controls PLK1 spatiotemporal regulation during mitosis

p53-binding protein 1 (53BP1) is a key mediator of the DNA damage response and genome stability. While its interphase function is well-characterized, its mitotic role remains less understood. Here we show that aberrant activation of 53BP1 through the loss of its negative regulator, TIRR, leads to mitotic abnormalities including altered spindle geometry, kinetochore-microtubule (k-MT) attachment errors and whole chromosome missegregation. We demonstrate that loss of TIRR results in excess interaction between 53BP1 and the key mitotic kinase Polo-like kinase 1 (PLK1), altering PLK1s activation, spatial distribution, and its interaction with known PLK1 substrates at multiple mitotic stages. Moreover, due to PLK1s established role in CENP-A loading, hyperactivation of 53BP1 compromises CENP-A loading, triggers gradual loss of CENP-A from centromeres and generates severe kinetochore assembly defects. These findings uncover a non-canonical mitotic function of 53BP1 as a key regulator of PLK1 activity and chromosome segregation fidelity.

Cell Biology↗

bioRxiv2026

Components of an ESCRT-independent nuclear envelope assembly pathway

In many cells, the nuclear envelope (NE) must be reassembled after mitosis 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 additional 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 interact and mutations disrupting the predicted interaction interface abolish Alx1s 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 mechanisms that maintain the integrity of the nucleus.

cell biology↗

bioRxiv2024

TTF2 promotes replisome eviction from stalled forks in mitosis

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 ATPase TTF2 is recruited to the replisome, where its non-catalytic N-terminal domain tethers Cyclin B-CDK1-phosphorylated TRAIP to the leading strand DNA polymerase {varepsilon} in a geometry that allows replisome ubiquitylation. Thus, a phospho-regulated architectural switch alters replisome organization in mitosis to safeguard genome integrity before chromosome segregation.

biochemistry↗

bioRxiv2024

Chromosome breakage-replication/fusion enables rapid DNA amplification

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.

cancer biology↗

bioRxiv2024

Haplotype-resolved karyotype construction from Hi-C data using refLinker

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 refLinkers broad utility for studying the functional consequences of chromosomal rearrangements.

genomics↗

bioRxiv2023

2D and 3D multiplexed subcellular profiling of nuclear instability in human cancer

Nuclear atypia, including altered nuclear size, contour, and chromatin organization, is ubiquitous in cancer cells. Atypical primary nuclei and micronuclei can rupture during interphase; however, the frequency, causes, and consequences of nuclear rupture are unknown in most cancers. We demonstrate that nuclear envelope rupture is surprisingly common in many human cancers, particularly glioblastoma. Using highly-multiplexed 2D and super-resolution 3D-imaging of glioblastoma tissues and patient-derived xenografts and cells, we link primary nuclear rupture with reduced lamin A/C and micronuclear rupture with reduced lamin B1. Moreover, ruptured glioblastoma cells activate cGAS-STING-signaling involved in innate immunity. We observe that local patterning of cell states influences tumor spatial organization and is linked to both lamin expression and rupture frequency, with neural-progenitor-cell-like states exhibiting the lowest lamin A/C levels and greatest susceptibility to primary nuclear rupture. Our study reveals that nuclear instability is a core feature of cancer, and links nuclear integrity, cell state, and immune signaling.

cancer biology↗

bioRxiv2022

Transgenerational transcriptional heterogeneity from cytoplasmic chromatin

Transcriptional heterogeneity from plasticity of the epigenetic state of chromatin is thought to contribute to tumor evolution, metastasis, and drug resistance 1-3. However, the mechanisms leading to nongenetic cell-to-cell variation in gene expression remain poorly understood. Here we demonstrate that heritable transcriptional changes can result from the formation of micronuclei, aberrations of the nucleus that are common in cancer4,5. Micronuclei have fragile nuclear envelopes (NE) that are prone to spontaneous rupture, which exposes chromosomes to the cytoplasm and disrupts many nuclear activities 6,7. Using a combination of long-term live-cell imaging and same-cell, single-cell RNA sequencing (Look-Seq2), we identified significant reduction of gene expression in micronuclei, both before and after NE rupture. Furthermore, chromosomes in micronuclei fail to normally recover histone 3 lysine 27 acetylation, a critical step for the reestablishment of normal transcription after mitosis 8-10. These transcription and chromatin defects can persist into the next generation in a subset of cells, even after these chromosomes are incorporated into normal daughter nuclei. Moreover, persistent transcriptional repression is strongly associated with, and may be explained by, surprisingly long-lived DNA damage to these reincorporated chromosomes. Therefore, heritable alterations in transcription can originate from aberrations of nuclear architecture.

cell biology↗

bioRxiv2021

Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

biophysics↗

bioRxiv2020

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3-6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures--micronuclei and chromosome bridges--that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

genetics↗

bioRxiv2019

Mechanisms Generating Cancer Genome Complexity From A Single Cell Division Error

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes with chromothripsis, another catastrophic mutational process. Here, we explain this association by identifying a mutational cascade downstream of chromosome bridge formation that generates increasing amounts of chromothripsis. We uncover a new role for actomyosin forces in bridge breakage and mutagenesis. Chromothripsis then accumulates starting with aberrant interphase replication of bridge DNA, followed by an unexpected burst of mitotic DNA replication, generating extensive DNA damage. Bridge formation also disrupts the centromeric epigenetic mark, leading to micronucleus formation that itself promotes chromothripsis. We show that this mutational cascade generates the continuing evolution and sub-clonal heterogeneity characteristic of many human cancers.

cancer biology↗

bioRxiv2018

Nuclear envelope assembly defects link mitotic errors to chromothripsis

Defects in the architecture or integrity of the nuclear envelope (NE) are associated with a variety of human diseases1. Micronuclei, one common nuclear aberration, are an origin for chromothripsis2,3, a catastrophic mutational process commonly observed in cancer genomes and other contexts4-6. Micronuclei have a defective NE, with the extensive chromosome fragmentation that generates chromothripsis occurring after abrupt, spontaneous loss of NE integrity7. After NE disruption, the exposed cytoplasmic DNA can additionally initiate proinflammatory signaling linked to senescence, metastasis, and the immune clearance of tumor cells8. Despite its broad physiological impact, the basis for the nuclear envelope fragility of micronuclei is unknown. Here we demonstrate that micronuclei undergo markedly defective NE assembly: Only \"core\" NE proteins9,10 assemble efficiently on lagging chromosomes whereas \"non-core\" NE proteins9,10, including nuclear pore complexes (NPCs), fail to properly assemble. Consequently, micronuclei have impaired nuclear import, and key nuclear proteins required to maintain the integrity of the NE and the genome fail to accumulate normally. We show that densely bundled spindle microtubules inhibit non-core NE assembly, leading to an irreversible NE assembly defect. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective NE assembly, prevent spontaneous NE disruption, and suppress DNA damage in micronuclei. Our findings indicate that chromosome segregation and NE assembly are only loosely coordinated through the timing of mitotic spindle disassembly. The absence of precise regulatory controls can explain why errors during mitotic exit are frequent, and a major trigger for catastrophic genome rearrangements5,6.

cell biology↗