Chromatin Proteins Research Articles

DEK oncoprotein participates in heterochromatin replication via SUMO-dependent nuclear bodies.

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis and stemness, but a characteristic oncogenic mechanism has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress, and delineates an avenue of further study for unraveling the contribution of DEK to cancer development.

Effects of Chromatin Structure Modifiers on the trans-Acting Heterochromatin Position Effect in Drosophila melanogaster.

The heterochromatin position effect is manifested in the inactivation of euchromatin genes transferred to heterochromatin. In chromosomal rearrangements, genes located near the new eu-heterochromatin boundary in the rearrangement (cis-inactivation) and, in rare cases, genes of a region of the normal chromosome homologous to the region of the eu-heterochromatin boundary of the chromosome with the rearrangement (trans-inactivation) are subject to inactivation. The In(2)A4 inversion is able to trans-inactivate the UAS-eGFP reporter gene located on the normal chromosome. We knockdown a number of chromatin proteins using temperature-controlled RNA interference and investigated the effect of knockdown on trans-inactivation of the reporter. We found suppression of trans-inactivation by knockdowns of Su(var)2-HP2, a protein that binds to the key heterochromatin protein HP1a, SAYP, a subunit of the chromatin remodelling complex, and Eggless histone methyltransferase (SETDB1), which introduces a H3K9me3 histone mark, recognized by the HP1a protein. The method of studying the effects of gene knockdown on heterochromatin position effects presented in this work is of independent methodological interest.

Updating nucleosome positions within individual genes using molecular modeling methods and mnase sequencing data

Organization of chromatin plays an important role in regulating the genetic machinery of the cell. The basic unit of chromatin packaging is a nucleosome, which harbors DNA of about 145 base pairs in length. The packaging of genetic material and its accessibility to transcription enzymes and other regulatory chromatin proteins depends on the positions of nucleosomes. MNase sequencing is used to examine nucleosome positions in a genome. MNase sequencing data are sufficient for detecting the presence of nucleosomes on the sequence, but a determination of the precise locations of nucleosomes can be problematic. Accurate determination of nucleosome positions requires additional data filtering and processing. In this study, using MNase sequencing data, a combined method based on geometric analysis of nucleosome chain molecular models is proposed for selecting possible nucleosome positions. The developed algorithm efficiently eliminates inaccessible nucleosome chain combinations and conformationally prohibited nucleosome positions.

Interactions of Nucleosomes with Acidic Patch-Binding Peptides: A Combined Structural Bioinformatics, Molecular Modeling, Fluorescence Polarization, and Single-Molecule FRET Study.

In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide-nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability.

HMGA2 directly mediates chromatin condensation in association with neuronal fate regulation

Identification of factors that regulate chromatin condensation is important for understanding of gene regulation. High-mobility group AT-hook (HMGA) proteins 1 and 2 are abundant nonhistone chromatin proteins that play a role in many biological processes including tissue stem-progenitor cell regulation, but the nature of their protein function remains unclear. Here we show that HMGA2 mediates direct condensation of polynucleosomes and forms droplets with nucleosomes. Consistently, most endogenous HMGA2 localized to transposase 5– and DNase I–inaccessible chromatin regions, and its binding was mostly associated with gene repression, in mouse embryonic neocortical cells. The AT-hook 1 domain was necessary for chromatin condensation by HMGA2 in vitro and in cellulo, and an HMGA2 mutant lacking this domain was defective in the ability to maintain neuronal progenitors in vivo. Intrinsically disordered regions of other proteins could substitute for the AT-hook 1 domain in promoting this biological function of HMGA2. Taken together, HMGA2 may regulate neural cell fate by its chromatin condensation activity.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same sidechain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Refined read-out: The hUHRF1 Tandem-Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3.

UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.

DNA condensation by cysteine containing tetra-arginine ligands as a model for ionizing radiation damage to chromatin

We examined DNA binding and condensation by AcCR4NH2 and CR4NH2 ligands. By this approach, we can model physical DNA damage by ionizing radiation. The binding constant was 1 × 106 L mol−1 at low ionic strength (ca. 10 mmol L−1 sodium ions) and the resulting condensate size was ca. 1 μm. Given the similar base pair concentrations of ca. 250 mmol L−1 in condensed DNA and chromatin, DNA damage yields by ionizing irradiation in condensates with related oligoarginine ligands are comparable to those in eukaryotic nuclei. Therefore, with the benefit of these results, we can envisage an experimental system suitable for modeling DNA damage by ionizing radiation because it reproduces well the essential conditions in cell nuclei that affect this damage, particularly the high concentrations of DNA and protein in chromatin and the resulting highly efficient radical scavenging environment.

Rice chromatin protein OsHMGB1 is involved in phosphate homeostasis and plant growth by affecting chromatin accessibility.

Although phosphorus is one of the most important essential elements for plant growth and development, the epigenetic regulation of inorganic phosphate (Pi) signaling is poorly understood. In this study, we investigated the biological function and mode of action of the high-mobility-group box 1 protein OsHMGB1 in rice (Oryza sativa), using molecular and genetic approaches. We determined that OsHMGB1 expression is induced by Pi starvation and encodes a nucleus-localized protein. Phenotypic analysis of Oshmgb1 mutant and OsHMGB1 overexpression transgenic plants showed that OsHMGB1 positively regulates Pi homeostasis and plant growth. Transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing indicated that OsHMGB1 regulates the expression of a series of phosphate starvation-responsive (PSR) genes by binding to their promoters. Furthermore, an assay for transposase-accessible chromatin followed by sequencing revealed that OsHMGB1 is involved in maintaining chromatin accessibility. Indeed, OsHMGB1 occupancy positively correlated with genome-wide chromatin accessibility and gene expression levels. Our results demonstrate that OsHMGB1 is a transcriptional facilitator that regulates the expression of a set of PSR genes to maintain Pi homeostasis in rice by increasing the chromatin accessibility, revealing a key epigenetic mechanism that fine-tune plant acclimation responses to Pi-limited environments.

Novel insights into bulky DNA damage formation and nucleotide excision repair from high-resolution genomics

DNA damages compromise cell function and fate. Cells of all organisms activate a global DNA damage response that includes a signaling stress response, activation of checkpoints, and recruitment of repair enzymes. Especially deleterious are bulky, helix-distorting damages that block transcription and replication. Due to their miscoding nature, these damages lead to mutations and cancer. In human cells, bulky DNA damages are repaired by nucleotide excision repair (NER). To date, the basic mechanism of NER in naked DNA is well defined. Still, there is a fundamental gap in our understanding of how repair is orchestrated despite the packaging of DNA in chromatin, and how it is coordinated with active transcription and replication. The last decade has brought forth huge advances in our ability to detect and assay bulky DNA damages and their repair at single nucleotide resolution across the human genome. Here we review recent findings on the effect of chromatin and DNA-binding proteins on the formation of bulky DNA damages, and novel insights on NER, provided by the recent application of genomic methods.

Chromosomal aberrations induced by Mitomycin C in canine lymphocytes

Chromosomal aberrations comprise an abnormal number of chromosomes as well as changes in the structure of the chromosomes. Aberrations in the structure of chromosomes, such as gaps and breaks in chromatids and chromosomes, acentric fragments, telomeric associations, deletions, early chromatid-separation and large scale effects, such as pulverized metaphases, and sticky metaphases. In this study, the effect of Mitomycin C, a recognized clastogen on human lymphocytes, was assayed in cultures of canine lymphocytes, an animal species that is flourishing in comparative medicine. Blood cultures were performed on samples from a male and a female dog. Cytotoxicity using the mitotic index and genotoxicity testing were performed with 0.25 µg/ml Mitomycin C. The total chromosomal aberrations were significantly higher due to the effect of Mitomycin C (P=0.0247). The number of chromatid breaks nearly quintupled, while pulverized metaphases were found to be six times more frequent, and endoduplicated cells were three times higher than in negative control cultures. The quantitatively most relevant chromosomal aberration was the presence of sticky metaphases, related to adverse effects in chromatin proteins. The potential use of canine lymphocytes for chromosomal aberration assay is emphasized in the evaluation or re-evaluation of the genotoxic in vitro effect of xenobiotics, to evidence chromosomal damage.

Neutrophil extracellular traps in intracerebral hemorrhage: implications for pathogenesis and therapeutic targets.

Intracerebral hemorrhage is a common neurological disease, and its pathological mechanism is complex. As the first recruited leukocyte subtype after intracerebral hemorrhage, neutrophils play an important role in tissue damage. In the past, it was considered that neutrophils performed their functions through phagocytosis, chemotaxis, and degranulation. In recent years, studies have found that neutrophils also have the function of secreting extracellular traps. Extracellular traps are fibrous structure composed of chromatin and granular proteins, which plays an important role in innate immunity. Studies have shown a large number of neutrophil extracellular traps in hematoma samples, plasma samples, and drainage samples after intracerebral hemorrhage. In this paper, we summarized the related mechanisms of neutrophil external traps and injury after intracerebral hemorrhage. Neutrophil extracellular traps are involved in the process of brain injury after intracerebral hemorrhage. The application of related inhibitors to inhibit the formation of neutrophil external traps or promote their dissolution can effectively alleviate the pathological damage caused by intracerebral hemorrhage.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Garcinol and Anacardic Acid, Natural Inhibitors of Histone Acetyltransferases, Inhibit Rhabdomyosarcoma Growth and Proliferation.

Rhabdomyosarcoma (RMS) is a malignant tumour of the soft tissues. There are two main histopathological types: alveolar and embryonal. RMS occurs mainly in childhood and is a result of the deregulation of growth and differentiation of muscle cell precursors. There is an increasing amount of data indicating that numerous epigenetic alterations within chromatin and histone proteins are involved in the pathogenesis of this malignancy. Histone acetylation is one of the most important epigenetic modifications that is catalysed by enzymes from the group of histone acetyltransferases (HAT). In this study, the impact of the natural histone acetyltransferase inhibitors (HATi)-garcinol (GAR) and anacardic acid (AA)-on the biology of RMS cells was evaluated through a series of in vitro tests measuring proliferation, viability, clonogenicity, cell cycle and apoptosis. Moreover, using oligonucleotide microarrays and real-time PCR, we identified several genes whose expression changed after GAR and AA treatment. The examined HATi significantly reduce the invasive phenotype of RMS cells by inhibiting the growth rate, viability and clonogenic abilities. What is more, these substances cause cell cycle arrest in the G2/M phase, induce apoptosis and affect the genetic expression of the endoplasmic reticulum stress sensors. GAR and AA may serve as promising potential anti-cancer drugs since they sensitize the RMS cells to chemotherapeutic treatment.

Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

BackgroundThe family with sequence similarity 20-member C (FAM20C) kinase, a Golgi casein kinase, which is responsible for phosphorylating the majority of the extracellular phosphoproteins within S-x-E/pS motifs, and is fundamentally associated with multiple biological processes to maintain cell proliferation, biomineralization, migration, adhesion, and phosphate homeostasis. In dissecting how FAM20C regulates downstream molecules and potential mechanisms, however, there are multiple target molecules of FAM20C, particularly many phenomena remain elusive, such as changes in cell-autonomous behaviors, incompatibility in genotypes and phenotypes, and others.MethodsHere, assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), proteomics, and phosphoproteomics were performed in Fam20c-dificient osteoblasts and to facilitate an integrated analysis and determine the impact of chromatin accessibility, genomic expression, protein alterations, signaling pathway, and post translational modifcations.ResultsBy combining ATAC-seq and RNA-seq, we identified TCF4 and Wnt signaling pathway as the key regulators in Fam20c-dificient cells. Further, we showed Calpastatin/Calpain proteolysis system as a novel target axis for FAM20C to regulate cell migration and F-actin cytoskeleton by integrated analysis of proteomics and phosphoproteomics. Furthermore, Calpastatin/Calpain proteolysis system could negatively regulate the Wnt signaling pathway.ConclusionThese observations implied that Fam20c knockout osteoblasts would cause cell homeostatic imbalance, involving changes in multiple signaling pathways in the conduction system.

The multifunctional protein HMGB1: 50 years of discovery.

Fifty years since the initial discovery of HMGB1 in 1973 as a structural protein of chromatin, HMGB1 is now known to regulate diverse biological processes depending on its subcellular or extracellular localization. These functions include promoting DNA damage repair in the nucleus, sensing nucleic acids and inducing innate immune responses and autophagy in the cytosol and binding protein partners in the extracellular environment and stimulating immunoreceptors. In addition, HMGB1 is a broad sensor of cellular stress that balances cell death and survival responses essential for cellular homeostasis and tissue maintenance. HMGB1 is also an important mediator secreted by immune cells that is involved in a range of pathological conditions, including infectious diseases, ischaemia-reperfusion injury, autoimmunity, cardiovascular and neurodegenerative diseases, metabolic disorders and cancer. In this Review, we discuss the signalling mechanisms, cellular functions and clinical relevance of HMGB1 and describe strategies to modify its release and biological activities in the setting of various diseases.

Abstract A052: Chromatin conformational changes in prostate cancer progression

Abstract Introduction. Prostate cancer (PCa) is the second most common cancer in men worldwide. Globally, it is among the top three leading causes of cancer-related mortality in men. Although many patients carry an indolent form of PCa, a subset of patients develops the aggressive form of PCa which is often treated by androgen depletion therapy. Despite treatment, the disease, in a subgroup of patients, progresses to develop castration resistant prostate cancer (CRPC). Despite the ongoing efforts, the underlying progression mechanisms are not yet fully understood. A more comprehensive understanding may offer translational benefits. Thus, using patient sample-derived high-throughput sequencing data from multiple epigenetic data layers together with expression levels of genes and proteins, we aim to uncover aberrant regulatory mechanisms and their role in PCa progression. Methods. We characterize the PCa progression-related conformational chromatin changes by applying Hi-C to clinical samples from a cohort of 18 patients, divided into three evenly-sized groups: benign prostatic hyperplasia (BPH), untreated prostate cancer (PC), and CRPC. We further characterize our findings by integrating chromatin accessibility, DNA methylation, gene and protein expression data from matching samples. Results. Looking at active and inactive (A/B) compartments, as inferred by Hi-C data, we observe that they are in high agreement across the sample groups. Integrating these A/B compartments with DNA-methylation data, we observe an elevated and decreased genome-wide methylation pattern respectively. Looking at topologically associating domain (TAD) boundaries, we observe a high agreement across sample groups as 70% of the TAD boundaries are shared by all groups. Focusing in on the chromatin loops, we observe more heterogeneity compared to the overall chromatin conformation as 38% of the loops are sample-specific. Detecting differential loops followed by their integration with gene expression data for genes overlapping the differential loop anchors, we observe an association between differential loop strength and gene expression as PC develops. Further integration of the differential loops with accessible chromatin regions, as inferred by ATAC-seq data, gene expression, and publicly available transcription factor (TF) binding data, we observe that distal enhancers of up-regulated genes in PC are enriched for PCa-relevant TFs such as FOXA1, HOXB13, and GATA2, but we see no enrichment for up-regulated loops in PC. In CRPC, these same TFs are enriched for up-regulated loops, but this does not seem to affect the gene expression. Conclusion. Our results suggest while PC samples undergo a more uniform set of chromatin conformational changes, CRPC samples indicate elevated heterogeneity in their acquired conformational changes potentially contributing to a more aberrant disease progression trajectory. Citation Format: Tuomo Virtanen, Ebrahim Afyounian, Riikka Nurminen, Teuvo Tammela, Alfonso Urbanucci, Juha Kesseli, Kirsi Rautajoki, Tapio Visakorpi, Matti Nykter. Chromatin conformational changes in prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A052.

Protocol of the Double-Click Seq Method to Evaluate the Deposition Bias of Chromatin Proteins.

Symmetrical deposition of parental and newly synthesized chromatin proteins over both sister chromatids is important for the maintenance of epigenetic integrity. However, the mechanisms to maintain equal distribution of parental and newly synthesized chromatid proteins over sister chromatids remains largely unknown. Here, we describe the protocol for the recently developed double-click seq method that enables mapping of asymmetry in the deposition of parental and newly synthesized chromatin proteins on both sister chromatids in DNA replication. The method involved metabolic labeling of new chromatin proteins with l-Azidohomoalanine (AHA) and newly synthesized DNA with Ethynyl-2'-deoxyuridine (EdU) followed by two subsequent click reactions for biotinylation and subsequently by corresponding separation steps. This enables isolation of parental DNA that was bound to nucleosomes containing new chromatin proteins. Sequencing of these DNA samples and mapping around origins of replication in the cellular DNA enables estimation of the asymmetry in deposition of chromatin proteins over the leading and lagging strand in DNA replication. Altogether, this method contributes to the toolbox to understand histone deposition in DNA replication. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metabolic labeling with AHA and EdU and isolation of nuclei Basic Protocol 2: First click reaction, MNase digestion and streptavidin enrichment of labeled nucleosomes Basic Protocol 3: Second click reaction, Replication-Enriched Nucleosome Sequencing (RENS) Protocol.

EXPLORING THE POTENTIAL CORRELATION BETWEEN NEUTROPHIL EXTRACELLULAR TRAPS AND PROGNOSIS IN PATIENTS WITH DIFFUSE LARGE B‐CELL LYMPHOMA AND HODGKIN’S LYMPHOMA

Introduction: Neutrophil extracellular traps (NETs) are web-like structures of decondensed chromatin and granule proteins that are released from neutrophils in response to infection. NET-release also occurs and contributes to inflammation, organ dysfunction and thrombosis in patients with non-infectious inflammatory conditions, such as cancer. In several solid cancer types, NETs have been associated with poorer prognosis by facilitating tumour growth and metastasis. One study has associated NETs with tumour progression and poorer prognosis in diffuse large B-cell lymphoma (DLBCL), however the knowledge of the role of NETs in lymphoma is sparse. Here, NETs were measured in plasma from patients with DLBCL and Hodgkin’s lymphoma (HL), and correlated to blood counts and clinical outcome of the patients. Methods: Plasma samples from patients with DLBCL (n = 72) and HL (n = 54), diagnosed between 2010 and 2015, were obtained from the Swedish Uppsala Umeå Comprehensive Cancer Consortium (U-CAN) biobank. The lymphoma samples, as well as healthy controls (n = 11), were analysed using a commercial enzyme-linked immunosorbent assay (ELISA) targeting citrullinated histone 3 in complex with externalized DNA, a biomarker considered specific for NETs. Data collected from patient records, including routine blood counts at diagnosis, clinical background and disease outcome, was correlated to the obtained plasma concentrations of NETs. Results: DLBCL and HL patients showed significantly elevated concentrations of NETs in plasma compared to healthy controls (median concentrations 60.07, 65.24 and 5.366 ng/mL, p < 0.0001). In both DLBCL and HL, trends towards better overall survival (p = 0.3308 and 0.0534 respectively) and better progression free survival (p = 0.2777 and 0.1056) were seen for patients with concentrations of NETs above median. While there was no correlation between sex or age and NET levels in DLBCL patients, HL patients under the age of 50 years showed a significant correlation with higher levels of NETs (p = 0.0021). In both DLBCL and HL patients, plasma NET-levels showed a positive correlation with the neutrophil count in blood at diagnosis (p = 0.0087 and 0.0001 respectively). A neutrophil-lymphocyte ratio above 3.0, a known predictor of worse outcome in cancer patients, showed a trend towards being correlated with worse prognosis in DLBCL patients, but this was less evident for HL patients. Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

Dictionary learning for integrative, multimodal and scalable single-cell analysis.

Mapping single-cell sequencing profiles to comprehensive reference datasets provides a powerful alternative to unsupervised analysis. However, most reference datasets are constructed from single-cell RNA-sequencing data and cannot be used to annotate datasets that do not measure gene expression. Here we introduce 'bridge integration', a method to integrate single-cell datasets across modalities using a multiomic dataset as a molecular bridge. Each cell in the multiomic dataset constitutes an element in a 'dictionary', which is used to reconstruct unimodal datasets and transform them into a shared space. Our procedure accurately integrates transcriptomic data with independent single-cell measurements of chromatin accessibility, histone modifications, DNA methylation and protein levels. Moreover, we demonstrate how dictionary learning can be combined with sketching techniques to improve computational scalability and harmonize 8.6 million human immune cell profiles from sequencing and mass cytometry experiments. Our approach, implemented in version 5 of our Seurat toolkit ( http://www.satijalab.org/seurat ), broadens the utility of single-cell reference datasets and facilitates comparisons across diverse molecular modalities.