Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Organization of Chromatin by Intrinsic and Regulated Phase Separation.

Eukaryotic chromatin is highly condensed but dynamically accessible to regulation and organized into subdomains. We demonstrate that reconstituted chromatin undergoes histone tail-driven liquid-liquid phase separation (LLPS) in physiologic salt and when microinjected into cell nuclei, producing dense and dynamic droplets. Linker histone H1 and internucleosome linker lengths shared across eukaryotes promote phase separation of chromatin, tune droplet properties, and coordinate to form condensates of consistent density in manners that parallel chromatin behavior in cells. Histone acetylation by p300 antagonizes chromatin phase separation, dissolving droplets in vitro and decreasing droplet formation in nuclei. In the presence of multi-bromodomain proteins, such as BRD4, highly acetylated chromatin forms a new phase-separated state with droplets of distinct physical properties, which can be immiscible with unmodified chromatin droplets, mimicking nuclear chromatin subdomains. Our data suggest a framework, based on intrinsic phase separation of the chromatin polymer, for understanding the organization and regulation of eukaryotic genomes.

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.

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.

Structural features of nucleosomes in interphase and metaphase chromosomes.

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0-1 and ±3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.

DNA Methylation and Histone H1 Jointly Repress Transposable Elements and Aberrant Intragenic Transcripts.

DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here, we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Hypomethylation of genes also activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.

Fuzzy Interactions Form and Shape the Histone Transport Complex.

Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In this study, we determined the cryo-EM structure of the Imp7:Impß:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impß, whereas the acidic loops of Impß and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impß dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions.

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.

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures.

Double-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila.

Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. Here we show that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. We also demonstrate that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. Our data suggest that cell cycle-dependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing.

HP1BP3, a Chromatin Retention Factor for Co-transcriptional MicroRNA Processing.

Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.

Linker Histone H1.4 Inhibits the Growth, Migration and EMT Process of Non-Small Cell Lung Cancer by Regulating ERK1/2 Expression.

H1.4 is one of the 11 variants of linker histone H1, and is associated with tumorigenesis and development of various cancers. However, it is unclear for the role of histone H1.4 in non-small cell lung cancer (NSCLC). In this study, we found that overexpression of H1.4 significantly inhibited the cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) processes, whereas silencing H1.4 by shRNA knockdown promoted these processes in NSCLC cell lines A549 and H1299. We further showed that H1.4 overexpression reduced ERK1/2 expression or its phosphorylation levels, while H1.4 knockdown increased ERK1/2 expression or phosphorylation levels in NSCLC. Furthermore, we demonstrated that H1.4 bound to the promoter of ERK1/2, and acted as a transcriptional suppressor to inhibit ERK1/2 expression in A549 or H1299 cells. Importantly, we found that ERK ecto-expression can largely recovered the inhibitory effects of H1.4 on cell viability, migration, invasion and EMT processes. In summary, our study reveals that the H1.4-ERK pathway is crucial for cell viability, migration, invasion and EMT of NSCLC and could be a therapeutic target for NSCLC.

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Histone H1.2 promotes hepatocarcinogenesis by regulating signal transducer and activator of transcription 3 signaling.

Linker histone H1.2 (H1.2), encoded by HIST1H1C (H1C), is a major H1 variant in somatic cells. Among five histone H1 somatic variants, upregulated H1.2 was found in human hepatocellular carcinoma (HCC) samples and in a diethylnitrosamine (DEN)-induced HCC mouse model. In vitro, H1.2 overexpression accelerated proliferation of HCC cell lines, whereas H1.2 knockdown (KD) had the opposite effect. In vivo, H1.2 insufficiency or deficiency (H1c KD or H1c KO) alleviated inflammatory response and HCC development in DEN-treated mice. Mechanistically, H1.2 regulated the activation of signal transducer and activator of transcription 3 (STAT3), which in turn positively regulated H1.2 expression by binding to its promoter. Moreover, upregulation of the H1.2/STAT3 axis was observed in human HCC samples, and was confirmed in mouse models of methionine-choline-deficient diet induced nonalcoholic steatohepatitis or lipopolysaccharide induced acute inflammatory liver injury. Disrupting this feed-forward loop by KD of STAT3 or treatment with STAT3 inhibitors rescued H1.2 overexpression-induced proliferation. Moreover, STAT3 inhibitor treatment-ameliorated H1.2 overexpression promoted xenograft tumor growth. Therefore, H1.2 plays a novel role in inflammatory response by regulating STAT3 activation in HCC, thus, blockade of the H1.2/STAT3 loop is a potential strategy against HCC.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Histone H1 eviction by the histone chaperone SET reduces cell survival following DNA damage.

Many chromatin remodeling and modifying proteins are involved in the DNA damage response, where they stimulate repair or induce DNA damage signaling. Interestingly, we identified that downregulation of the histone H1 (H1)-interacting protein SET results in increased resistance to a wide variety of DNA damaging agents. We found that this increased resistance does not result from alleviation of an inhibitory effect of SET on DNA repair but, rather, is the consequence of a suppressed apoptotic response to DNA damage. Furthermore, we provide evidence that the histone chaperone SET is responsible for the eviction of H1 from chromatin. Knockdown of H1 in SET-depleted cells resulted in re-sensitization of cells to DNA damage, suggesting that the increased DNA damage resistance in SET-depleted cells is the result of enhanced retention of H1 on chromatin. Finally, clonogenic survival assays showed that SET and p53 act epistatically in the attenuation of DNA damage-induced cell death. Taken together, our data indicate a role for SET in the DNA damage response as a regulator of cell survival following genotoxic stress.This article has an associated First Person interview with the first author of the paper.

Cloning and characterization of linker histone H1 gene in rohu, Labeo rohita.

Linker histone H1 (LHH1) is an abundant nuclear protein that condenses chromatin to form higher-order structure. The present study reported cloning and sequencing of 942 bp of LHH1 from liver tissue of rohu, Labeo rohita, with a complete coding sequence of 792 bp of having 263 amino acids. The phylogenetic tree of L. rohita LHH1 (LrLHH1) shared maximum similarity with that of Carassius auratus. The three dimensional model and domain architecture of LrLHH1 protein was also predicted using Swiss-Prot and SMART domain software. The expression of LHH1 during ontogeny showed significantly higher transcript level in milt, unfertilized eggs and up to 3 h post-fertilization followed by a dramatic decrease thereafter. The tissue-specific expression showed constitutive expression of LrLHH1 in all examined tissues. The expression of LHH1 during different infection models, namely, bacteria (Aeromonas hydrophila); ectoparasite (Argulus siamensis) and poly I:C induction revealed modulation in the level of expression at varied time points post-exposure in the liver and anterior kidney tissues of rohu. However, a synthetic peptide derived from LHH1 sequence of rohu did not have any detectable antibacterial activity. The present study provided necessary information on the role of this protein during ontogeny and innate immunity in Indian major carp species.

Interactome of Site-Specifically Acetylated Linker Histone H1.

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.