Biology subjects

Black, B. E.

Publications and source records attributed to Black, B. E..

18 recordsLinked to original sources

bioRxiv2025

A Population-Specific PARP1 Gene Variation Modulates PARP Trapping

Poly-(ADP-ribose) polymerase inhibitors (PARPi) block NAD+-binding pocket of PARP1, inhibiting PAR synthesis. However, they differ in their ability to retain PARP1 on damaged DNA to induce synthetic lethality in homologous recombination-deficient (HRD) cancer. Allosteric enzymatic activation requires destabilization of the helical domain (HD) of PARP1 and is indispensable for activation and chromatin retention induced by distinct PARPi. Here we report that the effect of the clinical PARPi talazoparib is robustly impacted by a common human polymorphism within the HD. PARP1V762 greatly enhanced talazoparib-driven allosteric retention on chromatin, prolonged XRCC1 recruitment, and enhanced cell killing. Talazoparib switches from Type-II PARPi behavior in PARP1A762 to allosteric, pro-retention Type-I behavior for PARP1V762. Thus, both PARPi efficacy and dose-limiting tolerability depends on PARP1 allele, motivating variant-guided cancer therapies. TEASEROne of the four FDA-approved PARPi drugs, talazoparib, is modulated by a PARP1 SNP that is widespread in the population.

cancer biology↗

bioRxiv2025

Rapid assembly of functional modules for generating human artificial chromosome constructs compatible with epigenetic centromere seeding

The ongoing development of human artificial chromosomes (HACs) will permit investigation into essential centromere processes and the means to deliver large genetic cargoes to target cells. Starting with large ([~]750 kb) yeast artificial chromosome (YAC)-based constructs limits the rampant multimerization that has complicated many prior types of HACs. Large YAC construction is accomplished using transformation-associated recombination (TAR) strategies that can become unwieldly when several functional modules are to be incorporated and tested. To address this issue, we developed an approach where modules are built using high-fidelity in vitro assembly strategies in a bacterial artificial chromosome (BAC) format. Then, the assembled modules are transferred in a simplified TAR step into a recipient YAC harboring the prokaryotic "stuffer" DNA that comprises a large portion of the final HAC construct. This approach is highly efficient with two-thirds of all screened yeast clones harboring the correct TAR product. Further, whole-genome Oxford Nanopore Technologies (ONT) sequencing/alignments, de novo assembly of the final YAC using a single ONT sequencing run, and close inspection of highly repetitive regions are all streamlined to rapidly validate clones that match the design. The fully sequenced, verified strain harboring a multi-module construct was then fused to human cells, where it efficiently formed functional HACs upon initial seeding with CENP-A-containing nucleosomes. We envision that the rapid assembly steps will be useful to quickly incorporate different functional modules, including diverse genetic cargoes, to engineer HACs with specific design features.

synthetic biology↗

bioRxiv2025

Young KRAB-zinc finger gene clusters are highly dynamic incubators of ERV-driven genetic heterogeneity in mice

KRAB-zinc finger proteins (KZFPs) comprise the largest family of mammalian transcription factors, rapidly evolving within and between species. Most KZFPs repress endogenous retroviruses (ERVs) and other retrotransposons, with KZFP gene numbers correlating with the ERV load across species, suggesting coevolution. How new KZFPs emerge in response to ERV invasions is currently unknown. Using a combination of long-read sequencing technologies and genome assembly, we present a first detailed comparative analysis of young KZFP gene clusters in the mouse lineage, which has undergone recent KZFP gene expansion and ERV infiltration. Detailed annotation of KZFP genes in a cluster on Mus musculus Chromosome 4 revealed parallel expansion and diversification of this locus in different mouse strains (C57BL/6J, 129S1/SvImJ and CAST/EiJ) and species (Mus spretus and Mus pahari). Our data supports a model by which new ERV integrations within young KZFP gene clusters likely promoted recombination events leading to the emergence of new KZFPs that repress them. At the same time, ERVs also increased their numbers by duplication instead of retrotransposition alone, unraveling a new mechanism for ERV enrichment at these loci.

genetics↗

bioRxiv2025

Solution conformational differences between conventional and CENP-A nucleosomes are accentuated by reversible deformation under high pressure

Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural -satellite DNA cloned from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone identity and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

biophysics↗

bioRxiv2024

Short RNA chaperones promote aggregation-resistant TDP-43 conformers to mitigate neurodegeneration

Aberrant aggregation of the prion-like, RNA-binding protein TDP-43 underlies several debilitating neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS). Here, we define how short, specific RNAs antagonize TDP-43 aggregation. Short, specific RNAs engage and stabilize the TDP-43 RNA-recognition motifs, which allosterically destabilizes a conserved helical region in the prion-like domain, thereby promoting aggregation-resistant conformers. By mining sequence space, we uncover short RNAs with enhanced activity against TDP-43 and diverse disease-linked variants. The solubilizing activity of enhanced short RNA chaperones corrects aberrant TDP-43 phenotypes in optogenetic models and ALS patient-derived neurons. Remarkably, an enhanced short RNA chaperone mitigates TDP-43 proteinopathy and neurodegeneration in mice. Our studies reveal mechanisms of short RNA chaperones and pave the way for the development of short RNA therapeutics for fatal TDP-43 proteinopathies.

biochemistry↗

bioRxiv2024

A PARP2-specific active site alpha-helix melts to permit DNA damage-induced enzymatic activation

PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site -helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site -helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between PARP1 and PARP2.

biochemistry↗

bioRxiv2024

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 m. 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.

biophysics↗

bioRxiv2023

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 Alzheimers 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.

pathology↗

bioRxiv2023

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. One-Sentence SummaryA quarter century after the first human artificial chromosomes, a solution to their uncontrolled multimerization is achieved.

molecular biology↗

bioRxiv2023

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.

biochemistry↗

bioRxiv2023

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

SUMMARYCentromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A1. 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 oocyte2,3, 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 Mis18, 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.

cell biology↗

bioRxiv2023

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 {pi}-satellite ({pi}-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 {pi}-sat-related repeat, {pi}-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. TeaserChromatin and kinetochore alterations arise in response to evolutionarily rapid changes to underlying repetitive centromere DNA.

genomics↗

bioRxiv2022

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.

biochemistry↗

bioRxiv2022

Equalizing epigenetically imprinted centromeres in early mammalian embryos

The CENP-A histone variant epigenetically defines centromeres, where its levels and locations are precisely maintained through mitotic cell divisions. However, differences in centromere CENP-A propagation in soma versus female/male germline remains poorly understood. Here, we generated CenpamScarlet mice and followed CENP-A dynamics in gametes, zygotes, and embryos. We found that, unlike somatic cells, progenitor female and male germ cells carry high centromeric CENP-A levels that decrease upon terminal differentiation. The reduction in CENP-A is differentially regulated between sexes, resulting in a ten-fold higher level in oocytes compared to sperm. In the zygote, the parent-of-origin CENP-A asymmetry is equalized prior to initial S-phase by redistribution of nuclear CENP-A from maternal to paternal chromosomes. Redistribution of CENP-A requires both CDK1/2 and PLK1 centromeric machinery. These experiments provide direct evidence for resetting of epigenetically imprinted centromeres in early pronuclear stage embryos and imply a mechanism to sense the non-equivalency of parental chromosomes. HighlightsO_LIIncreased CENP-A density at centromeres is a conserved property of germline stem cells while CENP-A reduction is coincident with germ cell differentiation C_LIO_LIPaternal and maternal CENP-A containing nucleosomes are intergenerationally inherited C_LIO_LICENP-A density at centromeres differs between male and female mature gametes C_LIO_LIUpon fertilization, maternal nuclear CENP-A is redistributed to equalize with parental CENP-A C_LIO_LICENP-C and MIS18BP1 are asymmetrically enriched in the parental pronuclei in accordance with CENP-A asymmetry. C_LIO_LILicensing for centromere equalization begins before zygotic DNA replication C_LI

developmental biology↗

bioRxiv2021

Molecular basis of UHRF1 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 or complexes. How the different protein functionalities of complex chromatin modulators are regulated is often unclear. Using a combination of biochemical, biophysical and structural approaches we delineated the regulation of the H3unmodified and H3K9me binding activities of the multidomain UHRF1 protein. The phosphoinositide PI5P interacts with two distant flexible linker regions of UHRF1 in a mode that is dependent on the polar head group and the acyl part of the phospholipid. The associated conformational rearrangements stably position the H3unmodified and H3K9me3 histone recognition modules of UHRF1 for multivalent and synergistic binding of the H3 tail. Our work highlights a novel 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.

biochemistry↗

bioRxiv2021

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADPribose modifications

Upon detecting DNA strand breaks, PARP1 and PARP2 produce the posttranslational modification poly(ADP-ribose) to orchestrate the cellular response to DNA damage. Histone PARylation factor 1 (HPF1) binds to PARP1/2 to directly regulate their catalytic output. HPF1 is required for the modification of serine residues with ADP-ribose, whereas glutamate/aspartate residues are modified in the absence of HPF1. PARP1 is an abundant nuclear protein, whereas HPF1 is present in much lower amounts, raising the question of whether HPF1 can pervasively modulate PARP1 activity. Here we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at the sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates and dissociates from multiple PARP1 molecules, initiating ADP-ribose modification of serine residues before modification can initiate on glutamate/aspartate residues. HPF1 accelerates the rate of attaching the first ADP-ribose, such that this initiation event is comparable to the rate of the elongation reaction to form poly(ADP-ribose). This "hit and run" mechanism ensures that HPF1 contributions to the PARP1/2 active site during initiation do not persist and interfere with PAR chain elongation at sites of DNA damage. HPF1 thereby balances initiation and elongation events to regulate PARP1/2 output. Structural analysis of HPF1 in complex with PARP1 provides first insights into the assembly on a DNA strand break, and the HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of the serine-ADP-ribose modification in cells and establish that HPF1 imparts the efficiency of serine-ADP-ribose modification required for an acute response to DNA damage.

biochemistry↗

bioRxiv2020

Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

Assembly of a functional kinetochore is critical for accurate chromosome segregation. Hierarchical recruitment of soluble components during kinetochore assembly is a highly regulated mitotic event, but the underlying steps are not well understood. In yeast and Xenopus egg extracts, soluble kinetochore components can spontaneously assemble into microtubule-binding subcomplexes. Although the molecular interactions among specific kinetochore components are evolutionary conserved in eukaryotes, it remains unclear which de novo assembly steps are permitted in extracts of mitotic human cells. By analyzing the recruitment of GFP-fused kinetochore proteins from human mitotic cell extracts to inner kinetochore components immobilized on microbeads, we reconstructed the interaction between CENP-C and CENP-A-containing nucleosomes. However, subsequent phospho-dependent binding of the Mis12 complex was less efficient, whereas binding of the Ndc80 complex was inhibited. Consistently, the microtubule-binding activity of native kinetochore components, as well as those assembled using a combination of native and recombinant human proteins, was weaker than that of recombinant Ndc80 complex alone. Such inhibitory mechanisms that prevent interactions between different kinetochore components are likely to guard against spurious formation of kinetochores in the cytosol of mitotic human cells, and imply existence of specific regulatory mechanisms that permit these interactions at the assembling kinetochore.

cell biology↗

bioRxiv2018

Hydrogen-deuterium exchange coupled to top- and middle-down mass spectrometry enables high-resolution measurements of histone tail dynamics before and after nucleosome assembly

Until recently, a major limitation of hydrogen deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization information was limited to the length of the peptide generated during proteolysis. Recently, however, it has been demonstrated that electron transfer dissociation (ETD) allows for preservation of deuterium label in the gas phase and therefore can be used to obtain more resolved information. To date, this technology has remained mostly limited to single, small, already well-characterized model proteins. Here, we optimize, expand, and adapt HDX-MS/MS capabilities to accommodate histone and nucleosomal complexes on top-down (TD) HDX-MS/MS and middle-down (MD) HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded rigorous analysis by traditional structural biology techniques for decades, revealing important novel insights into chromatin biology. This work represents the first heterogeneous protein complex and protein-DNA complex to be analyzed by TD- and MD-HDX-MS/MS, respectively. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.

biochemistry↗