Human Artificial Chromosomes that Bypass Centromeric DNA.

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere-the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts.

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 centromere propagation and the nature of CENP-a nucleosomes.

Centromeres direct chromosome inheritance, but in multicellular organisms their positions on chromosomes are primarily specified epigenetically rather than by a DNA sequence. The major candidate for the epigenetic mark is chromatin assembled with the histone H3 variant CENP-A. Recent studies offer conflicting evidence for the structure of CENP-A-containing chromatin, including the histone composition and handedness of the DNA wrapped around the histones. We present a model for the assembly and deposition of centromeric nucleosomes that couples these processes to the cell cycle. This model reconciles divergent data for CENP-A-containing nucleosomes and provides a basis for how centromere identity is stably inherited.

Rapid Detection and Signaling of DNA Damage by PARP-1.

Poly(ADP-ribosyl) polymerase-1 (PARP-1) is an abundant ADP-ribosyl transferase that regulates various biological processes. PARP-1 is widely recognized as a first-line responder molecule in DNA damage response (DDR). Here, we review the full cycle of detecting DNA damage by PARP-1, PARP-1 activation upon DNA binding, and PARP-1 release from a DNA break. We also discuss the allosteric consequence upon binding of PARP inhibitors (PARPi) and the opportunity to tune its release from a DNA break. It is now possible to harness this new understanding to design novel PARPi for treating diseases where cell toxicity caused by PARP-1 'trapping' on DNA is either the desired consequence or entirely counterproductive.

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly.

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

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-specific assembly of CENP-a nucleosomes is mediated by HJURP.

The centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from the chromatin assembly factors previously shown to load other histone H3 variants. Here we demonstrate that prenucleosomal CENP-A is complexed with histone H4, nucleophosmin 1, and HJURP. Recruitment of new CENP-A into nucleosomes at replicated centromeres is dependent on HJURP. Recognition by HJURP is mediated through the centromere targeting domain (CATD) of CENP-A, a region that we demonstrated previously to induce a unique conformational rigidity to both the subnucleosomal CENP-A heterotetramer and the corresponding assembled nucleosome. We propose HJURP to be a cell-cycle-regulated CENP-A-specific histone chaperone required for centromeric chromatin assembly.

The CENP-L-N Complex Forms a Critical Node in an Integrated Meshwork of Interactions at the Centromere-Kinetochore Interface.

During mitosis, the macromolecular kinetochore complex assembles on the centromere to orchestrate chromosome segregation. The properties and architecture of the 16-subunit Constitutive Centromere-Associated Network (CCAN) that allow it to build a robust platform for kinetochore assembly are poorly understood. Here, we use inducible CRISPR knockouts and biochemical reconstitutions to define the interactions between the human CCAN proteins. We find that the CCAN does not assemble as a linear hierarchy, and instead, each sub-complex requires multiple non-redundant interactions for its localization to centromeres and the structural integrity of the overall assembly. We demonstrate that the CENP-L-N complex plays a crucial role at the core of this assembly through interactions with CENP-C and CENP-H-I-K-M. Finally, we show that the CCAN is remodeled over the cell cycle such that sub-complexes depend on their interactions differentially. Thus, an interdependent meshwork within the CCAN underlies the centromere specificity and stability of the kinetochore.

PARP-1 Activation Requires Local Unfolding of an Autoinhibitory Domain.

Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD(+) binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.

A core viral protein binds host nucleosomes to sequester immune danger signals.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

The unique kind of human artificial chromosome: Bypassing the requirement for repetitive centromere DNA.

Centromeres are essential components of all eukaryotic chromosomes, including artificial/synthetic ones built in the laboratory. In humans, centromeres are typically located on repetitive α-satellite DNA, and these sequences are the "major ingredient" in first-generation human artificial chromosomes (HACs). Repetitive centromeric sequences present a major challenge for the design of synthetic mammalian chromosomes because they are difficult to synthesize, assemble, and characterize. Additionally, in most eukaryotes, centromeres are defined epigenetically. Here, we review the role of the genetic and epigenetic contributions to establishing centromere identity, highlighting recent work to hijack the epigenetic machinery to initiate centromere identity on a new generation of HACs built without α-satellite DNA. We also discuss the opportunities and challenges in developing useful unique sequence-based HACs.

Chaperone Nap1 shields histone surfaces used in a nucleosome and can put H2A-H2B in an unconventional tetrameric form.

The histone H2A-H2B heterodimer is an integral component of the nucleosome. The cellular localization and deposition of H2A-H2B into chromatin is regulated by numerous factors, including histone chaperones such as nucleosome assembly protein 1 (Nap1). We use hydrogen-deuterium exchange coupled to mass spectrometry to characterize H2A-H2B and Nap1. Unexpectedly, we find that at low ionic strength, the α helices in H2A-H2B are frequently sampling partially disordered conformations and that binding to Nap1 reduces this conformational sampling. We identify the interaction surface between H2A-H2B and Nap1 and confirm its relevance both in vitro and in vivo. We show that two copies of H2A-H2B bound to a Nap1 homodimer form a tetramer with contacts between H2B chains similar to those in the four-helix bundle structural motif. The organization of the complex reveals that Nap1 competes with histone-DNA and interhistone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.

The HIRA histone chaperone complex subunit UBN1 harbors H3/H4- and DNA-binding activity.

The HIRA histone chaperone complex is composed of the proteins HIRA, UBN1, and CABIN1 and cooperates with the histone chaperone ASF1a to specifically bind and deposit H3.3/H4 into chromatin. We recently reported that the UBN1 Hpc2-related domain (HRD) specifically binds to H3.3/H4 over H3.1/H4. However, the mechanism for HIRA complex deposition of H3.3/H4 into nucleosomes remains unclear. Here, we characterize a central region of UBN1 (UBN1 middle domain) that is evolutionarily conserved and predicted to have helical secondary structure. We report that the UBN1 middle domain has dimer formation activity and binds to H3/H4 in a manner that does not discriminate between H3.1 and H3.3. We additionally identify a nearby DNA-binding domain in UBN1, located between the UBN1 HRD and middle domain, which binds DNA through electrostatic contacts involving several conserved lysine residues. Together, these observations suggest a mechanism for HIRA-mediated H3.3/H4 deposition whereby UBN1 associates with DNA and dimerizes to mediate formation of an (H3.3/H4)2 heterotetramer prior to chromatin deposition.

The scaffolding protein JADE1 physically links the acetyltransferase subunit HBO1 with its histone H3-H4 substrate.

The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins in vitro to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions in vivo recapitulated these in vitro interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.

The new normal of structure/function studies in the era of CRISPR/Cas9.

Major advances in gene-editing technologies have enabled the rapid dissection of proteins in complex biological systems, facilitating biological experiments to complement biochemical studies with purified components. In this editorial, we highlight CRISPR/Cas9-based strategies to rapidly manipulate endogenous genes - strategies that have already transformed functional studies of proteins in metazoan systems. We further describe emerging tools using a catalytically dead version of Cas9 (dCas9) that do not cleave DNA, but can alter gene expression and/or local chromatin states, edit single nucleotide bases, and permit the visualization of specific genomic loci. Looking to the not-too-distant future, CRISPR/Cas9-based methodologies promise to lead to discoveries of new biology, opening the door for bold new synthetic biology platforms.

Centromere inheritance through the germline.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.

Identification of the Post-translational Modifications Present in Centromeric Chromatin.

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.

Cell Biology of Cheating-Transmission of Centromeres and Other Selfish Elements Through Asymmetric Meiosis.

Mendel's First Law of Genetics states that a pair of alleles segregates randomly during meiosis so that one copy of each is represented equally in gametes. Whereas male meiosis produces four equal sperm, in female meiosis only one cell, the egg, survives, and the others degenerate. Meiotic drive is a process in which a selfish DNA element exploits female meiotic asymmetry and segregates preferentially to the egg in violation of Mendel's First Law, thereby increasing its transmission to the offspring and frequency in a population. In principle, the selfish element can consist either of a centromere that increases its transmission via an altered kinetochore connection to the meiotic spindle or a centromere-like element that somehow bypasses the kinetochore altogether in doing so. There are now examples from eukaryotic model systems for both types of meiotic drive. Although meiotic drive has profound evolutionary consequences across many species, relatively little is known about the underlying mechanisms. We discuss examples in various systems and open questions about the underlying cell biology, and propose a mechanism to explain biased segregation in mammalian female meiosis.

A novel hybrid single molecule approach reveals spontaneous DNA motion in the nucleosome.

Structural dynamics of nucleic acid and protein is an important physical basis of their functions. These motions are often very difficult to synchronize and too fast to be clearly resolved with the currently available single molecule methods. Here we demonstrate a novel hybrid single molecule approach combining stochastic data analysis with fluorescence correlation that enables investigations of sub-ms unsynchronized structural dynamics of macromolecules. Based on the method, we report the first direct evidence of spontaneous DNA motions at the nucleosome termini. The nucleosome, comprising DNA and a histone core, is the fundamental packing unit of eukaryotic genes that must be accessed during various genome transactions. Spontaneous DNA opening at the nucleosome termini has long been hypothesized to enable gene access in the nucleosome, but has yet to be directly observed. Our approach reveals that DNA termini in the nucleosome open and close repeatedly at 0.1-1 ms(-1). The kinetics depends on salt concentration and DNA-histone interactions but not much on DNA sequence, suggesting that this dynamics is universal and imposes the kinetic limit to gene access. These results clearly demonstrate that our method provides an efficient and robust means to investigate unsynchronized structural changes of DNA at a sub-ms time resolution.

Molecular underpinnings of centromere identity and maintenance.

Centromeres direct faithful chromosome inheritance at cell division but are not defined by a conserved DNA sequence. Instead, a specialized form of chromatin containing the histone H3 variant, CENP-A, epigenetically specifies centromere location. We discuss current models where CENP-A serves as the marker for the centromere during the entire cell cycle in addition to generating the foundational chromatin for the kinetochore in mitosis. Recent elegant experiments have indicated that engineered arrays of CENP-A-containing nucleosomes are sufficient to serve as the site of kinetochore formation and for seeding centromeric chromatin that self-propagates through cell generations. Finally, recent structural and dynamic studies of CENP-A-containing histone complexes - before and after assembly into nucleosomes - provide models to explain underlying molecular mechanisms at the centromere.