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Promoters of developmental genes in embryonic stem cells (ESCs) are marked by histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in an asymmetric nucleosomal conformation, with each sister histone H3 carrying only one of the two marks. These bivalent domains are thought to poise genes for timely activation upon differentiation. Here, we show that asymmetric bivalent nucleosomes recruit repressive H3K27me3 binders but fail to enrich activating H3K4me3 binders, thereby promoting a poised state. Strikingly, the bivalent mark combination further promotes recruitment of specific chromatin proteins that are not recruited by each mark individually, including the lysine acetyltransferase (KAT) complex KAT6B. Knockout of KAT6B blocks neuronal differentiation, demonstrating that KAT6B is critical for proper bivalent gene expression during ESC differentiation. These findings reveal how readout of the bivalent histone marks directly promotes a poised state at developmental genes while highlighting how nucleosomal asymmetry is critical for histone mark readout and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localized kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gametes are produced via meiosis, a specialized cell division associated with frequent errors that cause birth defects and infertility. Uniquely in meiosis I, homologous chromosomes segregate to opposite poles, usually requiring their linkage by chiasmata, the products of crossover recombination. 1 The spindle checkpoint delays cell-cycle progression until all chromosomes are properly attached to microtubules, 2 but the steps leading to the capture and alignment of chromosomes on the meiosis I spindle remain poorly understood. In budding yeast meiosis I, Mad2 and Mad3 BUBR1 are equally important for spindle checkpoint delay, but biorientation of homologs on the meiosis I spindle requires Mad2, but not Mad3 BUBR1 . 3 , 4 Here we reveal the distinct functions of Mad2 and Mad3 BUBR1 in meiosis I chromosome segregation. Mad2 promotes the prophase to metaphase I transition, while Mad3 BUBR1 associates with the TOGL1 domain of Stu1 CLASP , a conserved plus-end microtubule protein that is important for chromosome capture onto the spindle. Homologous chromosome pairs that are proficient in crossover formation but fail to biorient rely on Mad3 BUBR1 -Stu1 CLASP to ensure their efficient attachment to microtubules and segregation during meiosis I. Furthermore, we show that Mad3 BUBR1 -Stu1 CLASP are essential to rescue the segregation of mini-chromosomes lacking crossovers. Our findings define a new pathway ensuring microtubule-dependent chromosome capture and demonstrate that spindle checkpoint proteins safeguard the fidelity of chromosome segregation both by actively promoting chromosome alignment and by delaying cell-cycle progression until this has occurred., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-A Cnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-A Cnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-A Cnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-A Cnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states., Competing Interests: Declaration of interests C.M.H. and A.G. are inventors on a patent covering the therapeutic targeting of TONSL for cancer therapy. A.G. is co-founder and chief scientific officer of Ankrin Therapeutics. A.G. is a member of Molecular Cell’s Scientific Advisory Board. A.I. and M.V.-A. are cofounders of EpiQMAx. G.M. is co-founder and member of the board of directors of Twelve Bio and is a member of the Scientific Advisory Board at Ensoma., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 CDK1 as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G 2 . 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones., Competing Interests: Declaration of interests C.M.H., D.J.P., H.H., and A.G. are inventors on a filed patent application covering the therapeutic targeting of TONSL for cancer therapy. A.G. is a co-founder and chief scientific officer (CSO) of Ankrin Therapeutics. A.G. is a member of Molecular Cell’s scientific advisory board., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19c CCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19c CCAN -dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Meiosis produces gametes through a specialized, two-step cell division, which is highly error prone in humans. Reductional meiosis I, where maternal and paternal chromosomes (homologs) segregate, is followed by equational meiosis II, where sister chromatids separate. Uniquely during meiosis I, sister kinetochores are monooriented and pericentromeric cohesin is protected. Here, we demonstrate that these key adaptations for reductional chromosome segregation are achieved through separable control of multiple kinases by the meiosis-I-specific budding yeast Spo13 protein. Recruitment of Polo kinase to kinetochores directs monoorientation, while independently, cohesin protection is achieved by containing the effects of cohesin kinases. Therefore, reductional chromosome segregation, the defining feature of meiosis, is established by multifaceted kinase control by a master regulator. The recent identification of Spo13 orthologs, fission yeast Moa1 and mouse MEIKIN, suggests that kinase coordination by a meiosis I regulator may be a general feature in the establishment of reductional chromosome segregation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and beta(2)-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell-cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate the engagement of more stable beta(2)-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s(-1) was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking dependence of doublet lifetime on shear rate was observed. At low shear (G = 14 s(-1)), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca(++)-depleted EDTA buffers. At G > or = 66 s(-1), doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G > or = 110 s(-1), led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s(-1). Partial inhibition of aggregate formation was achieved by blocking beta(2)-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.