Centromeric chromatin clearings demarcate the site of kinetochore formation.

The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. The kinetochore has been interrogated by electron microscopy since the middle of the last century, but with methodologies that compromised fine structure. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20-25 nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils that extend >1 µm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.

Structural basis for the phase separation of the chromosome passenger complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer's disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

Efficient formation of single-copy human artificial chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125-base pair DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. We describe an approach that efficiently forms single-copy HACs. It employs a ~750-kilobase construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks.

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.

Centromere innovations within a mouse species.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying centromere protein-A (CENP-A) nucleosomes at the nexus of a satellite repeat that we identified and termed π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 mega-base pairs of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance promotes accumulation of microtubule-binding components of the kinetochore and a microtubule-destabilizing kinesin of the inner centromere. We propose that the balance of pro- and anti-microtubule binding by the new centromere is what permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling.

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following chemical fixation, staining, and mechanical sectioning, which limit attainable resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) offers the potential to image unfixed cellular samples at higher resolution while preserving their native structures, but it requires samples to be frozen free from crystalline ice and thin enough to image via transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate the native ultrastructure of unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue directly on cryo-EM grids via plunge-freezing, as opposed to high pressure freezing which is generally used for thick samples. Following vitrification, we use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid. In comparison to gallium FIB, which is commonly used for biological samples, xenon plasma FIB is powerful enough to efficiently mill large volume samples, such as human brain tissue. Additionally, our approach allows for lamellae to be generated at variable depth inside the tissue as opposed to being limited to starting at the surface of the tissue. Lamellae generated in Alzheimer's disease brain tissue and imaged by cryo-ET reveal intact subcellular structures including components of autophagy and potential tau fibrils. Furthermore, we visualize myelin revealing intact compact myelin and functional cytoplasmic expansions such as cytoplasmic channels and the inner tongue. From these images we also measure the dimensions of myelin membranes, providing insight into how myelin basic protein forces out oligodendrocyte cytoplasm to form compact myelin and tightly links intracellular polar head groups of the oligodendrocyte plasma membrane. This approach provides a first view of unfixed, never previously frozen human brain tissue prepared by cryo-plasma FIB milling and imaged at high resolution by cryo-ET.

Efficient Formation of Single-copy Human Artificial Chromosomes.

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a ~750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly.

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A.1 In cultured somatic cells, an established paradigm of cell-cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germ line challenges this model because of the cell-cycle arrest between pre-meiotic S phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocytes,2,3 suggesting that a similar process may be required for centromere inheritance in mammals. To test this hypothesis, we developed an oocyte-specific conditional knockout (cKO) mouse for Mis18α, an essential component of the assembly machinery. We find that embryos derived from Mis18α knockout oocytes fail to assemble CENP-A nucleosomes prior to zygotic genome activation (ZGA), validating the knockout model. We show that deletion of Mis18α in the female germ line at the time of birth has no impact on centromeric CENP-A nucleosome abundance, even after 6-8 months of aging. In addition, there is no detectable detriment to fertility. Thus, centromere chromatin is maintained long-term, independent of new assembly during the extended prophase I arrest in mouse oocytes.

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly.

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A 1 . In cultured somatic cells, an established paradigm of cell cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germline challenges this model because of the cell cycle arrest between pre-meiotic S-phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocyte 2,3 , suggesting that a similar process may be required for centromere inheritance in mammals. However, we show that centromere chromatin is maintained long-term independent of new assembly during the extended prophase I arrest in mouse oocytes. Conditional knockout of Mis18α, an essential component of the assembly machinery, in the female germline at the time of birth has almost no impact on centromeric CENP-A nucleosome abundance nor any detectable detriment to fertility.

Structural Basis for the Phase Separation of the Chromosome Passenger Complex.

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium-exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Centromere Innovations Within a Mouse Species.

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying CENP-A nucleosomes at the nexus of a satellite repeat that we identified and term π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 Mbp of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance drives accumulation of microtubule-binding components of the kinetochore, as well as a microtubule-destabilizing kinesin of the inner centromere. The balance of pro- and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

The structural basis of the multi-step allosteric activation of Aurora B kinase.

Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography.

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P.

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus- and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Chromosomes: A nuclear neighborhood conducive to centromere formation.

Centromere identity is specified by nucleosomes containing the histone variant CENP-A. A new study reveals that subnuclear location dictates the efficiency with which a new centromere forms.

Something's gotta give at the centromeric chromatin foundation of the kinetochore.

Structures of the reconstituted human inner kinetochore complex by Pesenti et al. (2022) and Yatskevich et al. (2022) raise the question of whether it is the CENP-A nucleosome or the CCAN complex itself that provides the foundation for kinetochore assembly.

Epigenetic, genetic and maternal effects enable stable centromere inheritance.

Centromeres are defined epigenetically by the histone H3 variant CENP-A. The propagation cycle by which pre-existing CENP-A nucleosomes serve as templates for nascent assembly predicts the epigenetic memory of weakened centromeres. Using a mouse model with reduced levels of CENP-A nucleosomes, we find that an embryonic plastic phase precedes epigenetic memory through development. During this phase, nascent CENP-A nucleosome assembly depends on the maternal Cenpa genotype rather than the pre-existing template. Weakened centromeres are thus limited to a single generation, and parental epigenetic differences are eliminated by equal assembly on maternal and paternal centromeres. These differences persist, however, when the underlying DNA of parental centromeres differs in repeat abundance, as assembly during the plastic phase also depends on sufficient repetitive centromere DNA. With contributions of centromere DNA and the Cenpa maternal effect, we propose that centromere inheritance naturally minimizes fitness costs associated with weakened centromeres or epigenetic differences between parents.

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications.

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This "hit and run" mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub.

Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-AK124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub. Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations.