H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans. Histone citrullination strongly inhibits nucleosome stacking and self-association with a modest effect on chromatin folding, whereas the reconstituted arrays undergo a dramatic unfolding into open zigzag chains induced by histone citrullination. This study sheds light on the internal structure of compact chromatin nanoparticles and suggests a mechanism for how epigenetic changes in chromatin folding are retained across both open and condensed forms.

Structural mechanism of LIN28B nucleosome targeting by OCT4.

Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap ∼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.

Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography.

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

Distinct Structures and Dynamics of Chromatosomes with Different Human Linker Histone Isoforms.

The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1. There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive. Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197 bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin.

Extended and dynamic linker histone-DNA Interactions control chromatosome compaction.

Chromatosomes play a fundamental role in chromatin regulation, but a detailed understanding of their structure is lacking, partially due to their complex dynamics. Using single-molecule DNA unzipping with optical tweezers, we reveal that linker histone interactions with DNA are remarkably extended, with the C-terminal domain binding both DNA linkers as far as approximately ±140 bp from the dyad. In addition to a symmetrical compaction of the nucleosome core governed by globular domain contacts at the dyad, the C-terminal domain compacts the nucleosome's entry and exit. These interactions are dynamic, exhibit rapid binding and dissociation, are sensitive to phosphorylation of a specific residue, and are crucial to determining the symmetry of the chromatosome's core. Extensive unzipping of the linker DNA, which mimics its invasion by motor proteins, shifts H1 into an asymmetric, off-dyad configuration and triggers nucleosome decompaction, highlighting the plasticity of the chromatosome structure and its potential regulatory role.

The missing linker: emerging trends for H1 variant-specific functions.

Major advances in the chromatin and epigenetics fields have uncovered the importance of core histones, histone variants and their post-translational modifications (PTMs) in modulating chromatin structure. However, an acutely understudied related feature of chromatin structure is the role of linker histone H1. Previous assumptions of the functional redundancy of the 11 nonallelic H1 variants are contrasted by their strong evolutionary conservation, variability in their potential PTMs, and increased reports of their disparate functions, sub-nuclear localizations and unique expression patterns in different cell types. The commonly accepted notion that histone H1 functions solely in chromatin compaction and transcription repression is now being challenged by work from multiple groups. These studies highlight histone H1 variants as underappreciated facets of chromatin dynamics that function independently in various chromatin-based processes. In this review, we present notable findings involving the individual somatic H1 variants of which there are seven, underscoring their particular contributions to distinctly significant chromatin-related processes.

Centrosome linker diversity and its function in centrosome clustering and mitotic spindle formation.

The centrosome linker joins the two interphase centrosomes of a cell into one microtubule organizing center. Despite increasing knowledge on linker components, linker diversity in different cell types and their role in cells with supernumerary centrosomes remained unexplored. Here, we identified Ninein as a C-Nap1-anchored centrosome linker component that provides linker function in RPE1 cells while in HCT116 and U2OS cells, Ninein and Rootletin link centrosomes together. In interphase, overamplified centrosomes use the linker for centrosome clustering, where Rootletin gains centrosome linker function in RPE1 cells. Surprisingly, in cells with centrosome overamplification, C-Nap1 loss prolongs metaphase through persistent activation of the spindle assembly checkpoint indicated by BUB1 and MAD1 accumulation at kinetochores. In cells lacking C-Nap1, the reduction of microtubule nucleation at centrosomes and the delay in nuclear envelop rupture in prophase probably cause mitotic defects like multipolar spindle formation and chromosome mis-segregation. These defects are enhanced when the kinesin HSET, which normally clusters multiple centrosomes in mitosis, is partially inhibited indicating a functional interplay between C-Nap1 and centrosome clustering in mitosis.

Gene-Specific H1 Eviction through a Transcriptional Activator→p300→NAP1→H1 Pathway.

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a gene-specific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator→p300→NAP1→H1 pathway.

A DNA condensation code for linker histones.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Linker H1 is an 'epi'centre of plant defence.

The role of linker H1 histones in plant defence has recently been investigated. Sheikh et al. found that Arabidopsis thaliana plants that were lacking all three H1 proteins showed increased disease resistance, but when primed, failed to induce enhanced resistance. Differences in epigenetic patterns could be the cause of defective priming.

The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.

Multiscale chromatin dynamics and high entropy in plant iPSC ancestors.

Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

DiffPROTACs is a deep learning-based generator for proteolysis targeting chimeras.

PROteolysis TArgeting Chimeras (PROTACs) has recently emerged as a promising technology. However, the design of rational PROTACs, especially the linker component, remains challenging due to the absence of structure-activity relationships and experimental data. Leveraging the structural characteristics of PROTACs, fragment-based drug design (FBDD) provides a feasible approach for PROTAC research. Concurrently, artificial intelligence-generated content has attracted considerable attention, with diffusion models and Transformers emerging as indispensable tools in this field. In response, we present a new diffusion model, DiffPROTACs, harnessing the power of Transformers to learn and generate new PROTAC linkers based on given ligands. To introduce the essential inductive biases required for molecular generation, we propose the O(3) equivariant graph Transformer module, which augments Transformers with graph neural networks (GNNs), using Transformers to update nodes and GNNs to update the coordinates of PROTAC atoms. DiffPROTACs effectively competes with existing models and achieves comparable performance on two traditional FBDD datasets, ZINC and GEOM. To differentiate the molecular characteristics between PROTACs and traditional small molecules, we fine-tuned the model on our self-built PROTACs dataset, achieving a 93.86% validity rate for generated PROTACs. Additionally, we provide a generated PROTAC database for further research, which can be accessed at https://bailab.siais.shanghaitech.edu.cn/service/DiffPROTACs-generated.tgz. The corresponding code is available at https://github.com/Fenglei104/DiffPROTACs and the server is at https://bailab.siais.shanghaitech.edu.cn/services/diffprotacs.

Genome-wide Rules of Nucleosome Phasing in Drosophila.

Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

An antioxidant feedforward cycle coordinated by linker histone variant H1.2 and NRF2 that drives nonsmall cell lung cancer progression.

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.

Two C. elegans DM domain proteins, DMD-3 and MAB-3, function in late stages of male somatic gonad development.

To bring about sexual dimorphism in form, information from the sex determination pathway must trigger sex-specific modifications in developmental programs. DM-domain encoding genes have been found to be involved in sex determination in a multitude of animals, often at the level of male somatic gonad formation. Here we report our findings that the DM-domain transcription factors MAB-3 and DMD-3 function together in multiple steps during the late stages of C. elegans male somatic gonad development. Both mab-3 and dmd-3 are expressed in the linker cell and hindgut of L4 males and dmd-3 is also expressed in presumptive vas deferens cells. Furthermore, dmd-3, but not mab-3, expression in the linker cell is downstream of nhr-67, a nuclear hormone receptor that was previously shown to control late stages of linker cell migration. In mab-3; dmd-3 double mutant males, the last stage of linker cell migration is partially defective, resulting in aberrant linker cell shapes and often a failure of the linker cell to complete its migration to the hindgut. When mab-3; dmd-3 double mutant linker cells do complete their migration, they fail to be engulfed by the hindgut, indicating that dmd-3 and mab-3 activity are essential for this process. Furthermore, linker cell death and clearance are delayed in mab-3; dmd-3 double mutants, resulting in the linker cell persisting into adulthood. Finally, DMD-3 and MAB-3 function to activate expression of the bZIP transcription factor encoding gene zip-5 and downregulate the expression of the zinc metalloprotease ZMP-1 in the linker cell. Taken together, these results demonstrate a requirement for DM-domain transcription factors in controlling C. elegans male gonad formation, supporting the notion that the earliest DM-domain genes were involved in male somatic gonad development in the last common ancestor of the bilaterians.

The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

Different roles of the heterodimer architecture of galectin-4 in selective recognition of oligosaccharides and lipopolysaccharides having ABH antigens.

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.