ß-Glucan Reverses the Epigenetic State of LPS-Induced Immunological Tolerance.

Innate immune memory is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to microbial components such as lipopolysaccharide (LPS). We apply an integrated epigenomic approach to characterize the molecular events involved in LPS-induced tolerance in a time-dependent manner. Mechanistically, LPS-treated monocytes fail to accumulate active histone marks at promoter and enhancers of genes in the lipid metabolism and phagocytic pathways. Transcriptional inactivity in response to a second LPS exposure in tolerized macrophages is accompanied by failure to deposit active histone marks at promoters of tolerized genes. In contrast, ß-glucan partially reverses the LPS-induced tolerance in vitro. Importantly, ex vivo ß-glucan treatment of monocytes from volunteers with experimental endotoxemia re-instates their capacity for cytokine production. Tolerance is reversed at the level of distal element histone modification and transcriptional reactivation of otherwise unresponsive genes. VIDEO ABSTRACT.

Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

Molecular logic of cellular diversification in the mouse cerebral cortex.

The mammalian cerebral cortex has an unparalleled diversity of cell types, which are generated during development through a series of temporally orchestrated events that are under tight evolutionary constraint and are critical for proper cortical assembly and function1,2. However, the molecular logic that governs the establishment and organization of cortical cell types remains unknown, largely due to the large number of cell classes that undergo dynamic cell-state transitions over extended developmental timelines. Here we generate a comprehensive atlas of the developing mouse neocortex, using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing. We sampled the neocortex every day throughout embryonic corticogenesis and at early postnatal ages, and complemented the sequencing data with a spatial transcriptomics time course. We computationally reconstruct developmental trajectories across the diversity of cortical cell classes, and infer their spatial organization and the gene regulatory programs that accompany their lineage bifurcation decisions and differentiation trajectories. Finally, we demonstrate how this developmental map pinpoints the origin of lineage-specific developmental abnormalities that are linked to aberrant corticogenesis in mutant mice. The data provide a global picture of the regulatory mechanisms that govern cellular diversification in the neocortex.

Joint single-cell measurements of nuclear proteins and RNA in vivo.

Identifying gene-regulatory targets of nuclear proteins in tissues is a challenge. Here we describe intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq), a scalable method that measures multiplexed intranuclear protein levels and the transcriptome in parallel across thousands of nuclei, enabling joint analysis of transcription factor (TF) levels and gene expression in vivo. We apply inCITE-seq to characterize cell state-related changes upon pharmacological induction of neuronal activity in the mouse brain. Modeling gene expression as a linear combination of quantitative protein levels revealed genome-wide associations of each TF and recovered known gene targets. TF-associated genes were coexpressed as distinct modules that each reflected positive or negative TF levels, showing that our approach can disentangle relative putative contributions of TFs to gene expression and add interpretability to inferred gene networks. inCITE-seq can illuminate how combinations of nuclear proteins shape gene expression in native tissue contexts, with direct applications to solid or frozen tissues and clinical specimens.

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

The alarmin S100A9 hampers osteoclast differentiation from human circulating precursors by reducing the expression of RANK.

The alarmin S100A8/A9 is implicated in sterile inflammation-induced bone resorption and has been shown to increase the bone-resorptive capacity of mature osteoclasts. Here, we investigated the effects of S100A9 on osteoclast differentiation from human CD14+ circulating precursors. Hereto, human CD14+ monocytes were isolated and differentiated toward osteoclasts with M-CSF and receptor activator of NF-κB (RANK) ligand (RANKL) in the presence or absence of S100A9. Tartrate-resistant acid phosphatase staining showed that exposure to S100A9 during monocyte-to-osteoclast differentiation strongly decreased the numbers of multinucleated osteoclasts. This was underlined by a decreased resorption of a hydroxyapatite-like coating. The thus differentiated cells showed a high mRNA and protein production of proinflammatory factors after 16 h of exposure. In contrast, at d 4, the cells showed a decreased production of the osteoclast-promoting protein TNF-α. Interestingly, S100A9 exposure during the first 16 h of culture only was sufficient to reduce osteoclastogenesis. Using fluorescently labeled RANKL, we showed that, within this time frame, S100A9 inhibited the M-CSF-mediated induction of RANK. Chromatin immunoprecipitation showed that this was associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK was in part recovered by blocking TNF-α but not IL-1. Together, our data show that S100A9 impedes monocyte-to-osteoclast differentiation, probably via a reduction in RANK expression.-Di Ceglie, I., Blom, A. B., Davar, R., Logie, C., Martens, J. H. A., Habibi, E., Böttcher, L.-M., Roth, J., Vogl, T., Goodyear, C. S., van der Kraan, P. M., van Lent, P. L., van den Bosch, M. H. The alarmin S100A9 hampers osteoclast differentiation from human circulating precursors by reducing the expression of RANK.

Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome.

Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/- adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/- mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/- mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/- mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.

Expansion in situ genome sequencing links nuclear abnormalities to hotspots of aberrant euchromatin repression.

Microscopy and genomics are both used to characterize cell function, but approaches to connect the two types of information are lacking, particularly at subnuclear resolution. While emerging multiplexed imaging methods can simultaneously localize genomic regions and nuclear proteins, their ability to accurately measure DNA-protein interactions is constrained by the diffraction limit of optical microscopy. Here, we describe expansion in situ genome sequencing (ExIGS), a technology that enables sequencing of genomic DNA and superresolution localization of nuclear proteins in single cells. We applied ExIGS to fibroblast cells derived from an individual with Hutchinson-Gilford progeria syndrome to characterize how variation in nuclear morphology affects spatial chromatin organization. Using this data, we discovered that lamin abnormalities are linked to hotspots of aberrant euchromatin repression that may erode cell identity. Further, we show that lamin abnormalities heterogeneously increase the repressive environment of the nucleus in tissues and aged cells. These results demonstrate that ExIGS may serve as a generalizable platform for connecting nuclear abnormalities to changes in gene regulation across disease contexts.

Emerging roles of epigenetic mechanisms in Parkinson's disease.

Epigenetic mechanisms have emerged as important components of a variety of human diseases, including cancer and central nervous system disorders. Despite recent studies highlighting the role of epigenetic mechanisms in several neurodegenerative and neuropsychiatric disorders, to date, there has been a paucity of studies exploring the role of epigenetic factors in Parkinson's disease (PD). PD is a progressive neurological disorder with characteristic motor and non-motor symptoms, including a range of neuropsychiatric features, for which neither preventative nor effective long-term treatment strategies are available. It is one of the most common neurodegenerative disorders and the second most prevalent after Alzheimer's disease. In this review, we present several lines of evidence suggesting that epigenetic factors may play an important role in the pathogenesis of PD and propose on this basis a framework to guide future investigations into epigenetic mechanisms and systems biology of PD. These notions, together with technical advances in the ability to perform genome-wide analysis of epigenomic states, and newly available small-molecule probes targeting chromatin-modifying enzymes, may help design new treatment strategies for PD and other human diseases involving epigenetic dysregulation.

Compressed sensing for highly efficient imaging transcriptomics.

Recent methods for spatial imaging of tissue samples can identify up to ~100 individual proteins1-3 or RNAs4-10 at single-cell resolution. However, the number of proteins or genes that can be studied in these approaches is limited by long imaging times. Here we introduce Composite In Situ Imaging (CISI), a method that leverages structure in gene expression across both cells and tissues to limit the number of imaging cycles needed to obtain spatially resolved gene expression maps. CISI defines gene modules that can be detected using composite measurements from imaging probes for subsets of genes. The data are then decompressed to recover expression values for individual genes. CISI further reduces imaging time by not relying on spot-level resolution, enabling lower magnification acquisition, and is overall about 500-fold more efficient than current methods. Applying CISI to 12 mouse brain sections, we accurately recovered the spatial abundance of 37 individual genes from 11 composite measurements covering 180 mm2 and 476,276 cells.

Publisher Correction: Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

In the version of the article originally published, extra lines were displayed in Fig. 7. Fig. 7a contained a solid black line that extended into panel b, and Fig. 7c contained two extra scale bars on the left. These have been removed from the figure. The errors have been corrected in the HTML and PDF versions of the article.

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

Chromatin-Based Classification of Genetically Heterogeneous AMLs into Two Distinct Subtypes with Diverse Stemness Phenotypes.

Global investigation of histone marks in acute myeloid leukemia (AML) remains limited. Analyses of 38 AML samples through integrated transcriptional and chromatin mark analysis exposes 2 major subtypes. One subtype is dominated by patients with NPM1 mutations or MLL-fusion genes, shows activation of the regulatory pathways involving HOX-family genes as targets, and displays high self-renewal capacity and stemness. The second subtype is enriched for RUNX1 or spliceosome mutations, suggesting potential interplay between the 2 aberrations, and mainly depends on IRF family regulators. Cellular consequences in prognosis predict a relatively worse outcome for the first subtype. Our integrated profiling establishes a rich resource to probe AML subtypes on the basis of expression and chromatin data.

Multi-omics profiling reveals a distinctive epigenome signature for high-risk acute promyelocytic leukemia.

Epigenomic alterations have been associated with both pathogenesis and progression of cancer. Here, we analyzed the epigenome of two high-risk APL (hrAPL) patients and compared it to non-high-risk APL cases. Despite the lack of common genetic signatures, we found that human hrAPL blasts from patients with extremely poor prognosis display specific patterns of histone H3 acetylation, specifically hyperacetylation at a common set of enhancer regions. In addition, unique profiles of the repressive marks H3K27me3 and DNA methylation were exposed in high-risk APLs. Epigenetic comparison with low/intermediate-risk APLs and AMLs revealed hrAPL-specific patterns of histone acetylation and DNA methylation, suggesting these could be further developed into markers for clinical identification. The epigenetic drug MC2884, a newly generated general HAT/EZH2 inhibitor, induces apoptosis of high-risk APL blasts and reshapes their epigenomes by targeting both active and repressive marks. Together, our analysis uncovers distinctive epigenome signatures of hrAPL patients, and provides proof of concept for use of epigenome profiling coupled to epigenetic drugs to 'personalize' precision medicine.

Dynamics of Transcription Regulation in Human Bone Marrow Myeloid Differentiation to Mature Blood Neutrophils.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

Transcriptional and epigenetic control in mouse pluripotency: lessons from in vivo and in vitro studies.

Pluripotent cells were first derived from mouse blastocysts several decades ago. Since then, our knowledge of the molecular events that occur in the pre-implantation embryo has been vastly progressing. The emergence of epigenetics has revolutionized stem cell and developmental biology and further deepened our understanding of the underlying molecular mechanisms controlling the early embryo development. In particular, the emergence of massive parallel sequencing technologies has opened new avenues and became indispensable tools in modern biology. Additionally, development of new and exciting techniques for genome manipulation (TALEN and CRISPR/Cas9) and in vivo imaging provide unique opportunities to perturb and trace biological systems at very high resolution. Finally, recent single-cell - omics combined with sophisticated computational methodologies allow accurate, quantitative measurements for deconvolution of cellular variation in complex cell populations. Collectively, these achievements enabled the detailed characterization and monitoring of various cell states and trajectories during early stages of embryonic development. Here we review recent studies of the transcriptional and epigenetic changes during very early stages of mouse embryo development and compare these with pluripotent cells grown in vitro under different culture conditions. We discuss whether the in vitro cell states have an 'epi-phenocopy' in the embryo and refine our understanding of the circuitries controlling pluripotency and lineage commitment during early stages of mouse development.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor ß signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity.

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by ß-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to ß-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by ß-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.