Biology subjects

Earnshaw, W. C.

Publications and source records attributed to Earnshaw, W. C..

12 recordsLinked to original sources

bioRxiv2026

Molecular Mechanism of Condensin II auto-repression by NCAPD3 and activation by M18BP1

Human condensin II is a constitutively nuclear molecular motor that initiates chromosome organization in early mitosis. How condensin II is activated specifically in mitosis remains unknown. Here, we describe the molecular mechanism underlying condensin II auto-repression and activation. By determining multiple structural states of condensin II, we discovered that an autoinhibitory tail within the NCAPD3 subunit (NCAPD3Tail) holds the complex in a conformation that is incompatible with DNA capture. Deletion of NCAPD3Tail spontaneously activates condensin II in cells, illuminating its autoinhibitory role in vivo. We further show this translates to increased loop DNA formation by condensin II in vitro. Direct competition for the NCAPD3Tail binding site on condensin II enables the putative activator protein M18BP1 to liberate a key DNA-binding element in the NCAPH2 N-terminus, enabling DNA capture. Unexpectedly, M18BP1 not only relieves autoinhibition but also directly contributes to DNA organization by forming a positively charged loop that enhances DNA-anchoring by condensin II. Together, these findings reveal a bipartite activation mechanism wherein M18BP1 relieves autorepression, and renders condensin II biochemically competent to form stable DNA loops, ensuring highly stringent regulation of mitotic chromosome formation.

biochemistry↗

bioRxiv2025

A time-resolved atlas of histone modifications during mitotic entry

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms--particularly those involving phosphorylation and deacetylation of specific histone residues--have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

cell biology↗

bioRxiv2025

Calyculin A Induces Premature Chromosome Condensation and Chromatin Compaction in G1-Phase HeLa Cells without Histone H1 Phosphorylation

We show here that treatment of HeLa cells with calyculin A, an inhibitor of Protein Phosphatases 1 and 2A, induces premature chromosome condensation (PCC) at any point in interphase of the cell cycle. Chromosomes in G1-phase PCC closely resemble metaphase chromatids in the light microscope, and measurements using FLIM-FRET show that they have the same level of chromatin compaction as metaphase chromosomes. However, histone H1 is not phosphorylated in G1- or early S-phase PCC. These results suggest that H1 phosphorylation is not required for mitotic chromosome condensation and chromatin compaction. They also confirm that Cdk1/cyclin B, which directly phosphorylates histone H1, is not active in G1 and thus is not essential for G1- PCC. We suggest that induction of G1-PCC involves protein kinases or other factors that are either held in an inactive state by protein phosphatases, or constitutively active but countered by phosphatases. The same factors may be involved in the onset of normal mitosis, becoming active when protein phosphatases are downregulated. Induction of PCC with calyculin A should provide a useful system for identifying and studying the biochemical pathways that are required for mitotic chromosome compaction, nuclear envelope breakdown, and other events of mitosis.

cell biology↗

bioRxiv2025

Organisation of axial regions of isolated mitotic chromosomes visualised by cryo correlative light and electron tomography

The formation of mitotic chromosomes is essential for the accurate segregation of genetic material during cell division. Increasing evidence suggests that chromosome formation involves the reorganization of DNA into loops anchored within chromosomal axial regions, whose structural organization remains insufficiently characterized. Taking advantage of DT40 cells, an avian cell model characterized by the presence of a range of chromosome sizes from 3.2-197 Mb, we have established a preparation of entire close-to-native native mitotic chromosomes for cryogenic correlative light and electron microscopy (cryo-CLEM). The size of the smallest chromosomes allows imaging of their axial regions without further thinning. Cryo-electron tomography of the chromosome axial regions reveals the presence of heterogeneous non-histone macromolecular densities (NHMDs), approximately 30-45 nm in size, interspersed within chromatin/DNA regions. We propose that NHMDs may contain condensins and contribute to chromosome architecture. In addition to NHMDs, we identified dense clusters of particles, similar in size, near the chromosome surface, likely associated with ribosomal components. To quantitatively differentiate NHMDs from these surface clusters, we developed an analytical approach based on particle interspacing and spatial distribution within the chromosome volume. By establishing a cryo-CLEM workflow for whole, near-native mitotic chromosomes, our study provides a foundation for investigating their ultrastructural architecture.

cell biology↗

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2023

Kinetochore dynein is sufficient to biorient chromosomes and remodel the outer kinetochore

Multiple microtubule-directed activities concentrate on chromosomes during mitosis to ensure their accurate distribution to daughter cells. These activities include couplers and dynamics regulators localized at the kinetochore, the specialized microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and to mitotic chromatin. Here, we describe an in vivo reconstruction approach in which the effect of removing the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. This approach revealed that the kinetochore dynein module, comprised of the minus end-directed motor cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes and to remodel outer kinetochore composition following microtubule attachment; by contrast, the kinetochore dynein module is unable to support chromosome congression. The chromosome-autonomous action of kinetochore dynein, in the absence of the other major microtubule-directed factors on chromosomes, rotates and orients a substantial proportion of chromosomes such that their sister chromatids attach to opposite spindle poles. In tight coupling with orientation, the kinetochore dynein module drives removal of outermost kinetochore components, including the dynein motor itself and spindle checkpoint activators. The removal is independent of the other major microtubule-directed activities and kinetochore-localized protein phosphatase 1, suggesting that it is intrinsic to the kinetochore dynein module. These observations indicate that the kinetochore dynein module has the ability coordinate chromosome biorientation with attachment state-sensitive remodeling of the outer kinetochore that facilitates cell cycle progression.

cell biology↗

bioRxiv2022

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer cells.

cell biology↗

bioRxiv2021

Mapping the invisible chromatin transactions of prophase chromosome remodelling

We have used a combination of chemical genetics, chromatin proteomics and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 minutes following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.

cell biology↗

bioRxiv2020

Analysis of Complex DNA Rearrangements During Early Stages of HAC Formation

Human Artificial Chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN) and possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

synthetic biology↗

bioRxiv2019

Direct Nucleosome Binding of Borealin Secures Chromosome Association and Function of the Chromosomal Passenger complex

Chromosome association of the Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres [1-3] failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.

cell biology↗

bioRxiv2017

Mitotic chromosomes fold by condensin-dependent helical winding of chromatin loop arrays

During mitosis, chromosomes fold into compacted rod shaped structures. We combined imaging and Hi-C of synchronous DT40 cell cultures with polymer simulations to determine how interphase chromosomes are converted into compressed arrays of loops characteristic of mitotic chromosomes. We found that the interphase organization is disassembled within minutes of prophase entry and by late prophase chromosomes are already folded as arrays of consecutive loops. During prometaphase, this array reorganizes to form a helical arrangement of nested loops. Polymer simulations reveal that Hi-C data are inconsistent with solenoidal coiling of the entire chromatid, but instead suggest a centrally located helically twisted axis from which consecutive loops emanate as in a spiral staircase. Chromosomes subsequently shorten through progressive helical winding, with the numbers of loops per turn increasing so that the size of a helical turn grows from around 3 Mb (~40 loops) to ~12 Mb (~150 loops) in fully condensed metaphase chromosomes. Condensin is essential to disassemble the interphase chromatin conformation. Analysis of mutants revealed differing roles for condensin I and II during these processes. Either condensin can mediate formation of loop arrays. However, condensin II was required for helical winding during prometaphase, whereas condensin I modulated the size and arrangement of loops inside the helical turns. These observations identify a mitotic chromosome morphogenesis pathway in which folding of linear loop arrays produces long thin chromosomes during prophase that then shorten by progressive growth of loops and helical winding during prometaphase.\n\nOne Sentence SummaryMitotic chromosome morphogenesis occurs through condensin-mediated disassembly of the interphase conformation and formation of extended prophase loop arrays that then shorten by loop growth and condensin-dependent helical winding.

genomics↗

bioRxiv2017

Rapid degradation and 3D CLEM of condensin reveal chromatin compaction uncoupled from chromosome architecture in mitosis

The requirement for condensin in chromosome formation in somatic cells remains unclear as imperfectly condensed chromosomes do form in conventional condensindepleted cells. Here we have dissected the role of condensin at different stages of vertebrate mitosis by combining auxin-mediated rapid depletion of condensin subunit SMC2 with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells. We analysed the outcomes by live and fixed-cell microscopy methods, including 3D correlative light and serial block face scanning electron microscopy. Following rapid depletion of condensin, chromosomal defects were obvious. The chromatin was compacted normally, but formed a single mass of mitotic chromosomes clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments titrating the auxin concentration suggest a previously unsuspected dual role of condensin, as different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.

cell biology↗