Biology subjects

Kelly, D. A.

Publications and source records attributed to Kelly, D. A..

11 recordsLinked to original sources

bioRxiv2024

PLK1-Mediated Phosphorylation Cascade Activates the Mis18 Complex to Ensure Centromere Inheritance

Accurate chromosome segregation requires the attachment of spindle microtubules to centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cell-cycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 interacts with Mis18 and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18 (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18 and subsequent PLK1 binding is required to activate the Mis18/{beta} complex for robust Mis18/{beta}-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance. One-Sentence SummaryPLK1 phosphorylation cascade licenses CENP-A loading by facilitating HJURP centromere recruitment via Mis18/{beta} activation.

cell biology↗

bioRxiv2023

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which can interbreed with Mus musculus but lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

cell biology↗

bioRxiv2023

Age-dependent loss of cohesion protection in human oocytes

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage and chromosomal disorders. Such aneuploidies increase greatly as women age, originating from defective linkages between sister-chromatids (cohesion) in meiosis. We found evidence that loss of a specific pool of the cohesin protector protein, shugoshin 2 (Sgo2) contributes to this phenomenon. Our data indicate that Sgo2 preserves sister chromatid cohesion in meiosis by protecting a cohesin bridge between sister chromatids. In human oocytes, Sgo2 localizes to both sub-centromere cups and the pericentromeric bridge which spans the sister chromatid junction. Sgo2 normally colocalizes with cohesin, however, in oocytes from older women, Sgo2 is frequently lost specifically from the pericentromeric bridge and sister chromatid cohesion is weakened. Mps1 and Bub1 kinase activities maintain Sgo2 at sub-centromeres and the pericentromeric bridge. Removal of Sgo2 throughout meiosis I by Mps1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, Sgo2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that maternal age-dependent loss of Sgo2 at the pericentromere bridge in human oocytes impairs cohesion integrity and contributes to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

cell biology↗

bioRxiv2021

Centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

cell biology↗

bioRxiv2021

Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPCs intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated kinetochore proximal centromere pool of CPC.

cell biology↗

bioRxiv2020

Microtubule-independent movement of the fission yeast nucleus

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

cell biology↗

bioRxiv2020

The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19CCAN complex in meiotic kinetochore assembly

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganisation of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that replace the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that this kinetochore remodelling in meiosis requires the Ctf19cCCAN inner kinetochore complex. Through functional analyses, we identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth, but becomes critical upon meiotic entry. Therefore, extensive kinetochore remodelling and a distinct assembly pathway direct the specialization of meiotic kinetochores for successful gametogenesis.

cell biology↗

bioRxiv2019

SUMOylation targets shugoshin to stabilize sister kinetochore biorientation

The accurate segregation of chromosomes during mitosis relies on the attachment of sister chromatids to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule remains unclear. Here we identify SUMOylation as a mechanism that promotes anaphase onset upon biorientation. SUMO ligases modify the tension-sensing pericentromere-localized chromatin protein, shugoshin, to stabilize bioriented sister kinetochore-microtubule attachments. In the absence of SUMOylation, Aurora B kinase removal from kinetochores is delayed. Shugoshin SUMOylation prevents its binding to protein phosphatase 2A (PP2A) and release of this interaction is important for stabilizing sister kinetochore biorientation. We propose that SUMOylation modulates the kinase-phosphatase balance within pericentromeres to inactivate the error correction machinery, thereby allowing anaphase entry in response to biorientation.

cell 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↗

bioRxiv2019

Convergent genes shape budding yeast pericentromeres

The 3D architecture of the genome governs its maintenance, expression and transmission. The conserved ring-shaped cohesin complex organises the genome by topologically linking distant loci on either a single DNA molecule or, after DNA replication, on separate sister chromatids to provide the cohesion that resists the pulling forces of spindle microtubules during mitosis1,2. Cohesin is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres3-7. However, the structural organisation of pericentromeres and implications for chromosome segregation are unknown. Here we report the 3D structure of budding yeast pericentromeres and establish the relationship between genome organisation and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organised into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Re-orienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation in mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading at centromeres shapes pericentromeres into a structure competent for chromosome segregation during mitosis. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded and the re-structuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between 3D genome organization of a specific chromosomal domain and cellular function.

cell biology↗

bioRxiv2018

Spo13 prevents premature cohesin cleavage during meiosis

Meiosis produces gametes through two successive nuclear divisions, meiosis I and meiosis II. In contrast to mitosis and meiosis II, where sister chromatids are segregated, during meiosis I, homologous chromosomes are segregated. This requires the monopolar attachment of sister kinetochores and the loss of cohesion from chromosome arms, but not centromeres, during meiosis I. The establishment of both sister kinetochore mono-orientation and cohesion protection rely on the budding yeast meiosis I-specific Spo13 protein, the functional homolog of fission yeast Moa1 and mouse MEIKIN. Here we investigate the effects of loss of SPO13 on cohesion during meiosis I. Unlike wild type, cells lacking SPO13 fail to maintain the meiosis-specific cohesin subunit, Rec8, at centromeres and segregate sister chromatids to opposite poles during anaphase I. We show that the cohesin-destabilizing factor, Wpl1, is not primarily responsible for the loss of cohesion during meiosis I. Instead, premature loss of centromeric cohesin during anaphase I in spo13{Delta} cells relies on separase-dependent cohesin cleavage. Further, cohesin loss in spo13{Delta} anaphase I cells is blocked by forcibly tethering the regulatory subunit of protein phosphatase 2A, Rts1, to Rec8. Our findings indicate that separase-dependent cleavage of phosphorylated Rec8 causes premature cohesin loss in spo13{Delta} cells.

cell biology↗