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.

We Can Still Be Friends: IFN-γ Breaks Up Macrophage Enhancers.

Interferon (IFN)-γ can prime macrophages for inflammatory responses by several mechanisms, including enhancer establishment and gene activation. In this issue of Immunity, Kang et al. (2017) provide insight into the mechanisms of IFN-γ-mediated gene repression as they show that IFN-γ promotes the disassembly of select active enhancers by interfering with enhancer-binding transcription factor MAF.

Transcriptomic profiling of osteoarthritis synovial macrophages reveals a tolerized phenotype compounded by a weak corticosteroid response.

OBJECTIVES: It is well-known that long-term osteoarthritis prognosis is not improved by corticosteroid treatments. Here we investigate what could underlie this phenomenon by measuring the short term corticosteroid response of OA-Mf. METHODS: We determined the genome-wide transcriptomic response to corticosteroids of end-stage osteoarthritic joint synovial macrophages (OA-Mf). This was compared with LPS-tolerized and ß-glucan-trained circulating blood monocyte-derived macrophage models. RESULTS: Upon corticosteroid stimulation, the trained and tolerized macrophages significantly alter the abundance of 201 and 257 RNA transcripts, respectively. By contrast, by the same criteria, OA-Mf have a very restricted corticosteroid response of only 12 RNA transcripts. Furthermore, while metalloproteinases 1, -2, -3 and -10 expression clearly distinguish OA-Mf from both the tolerized and trained macrophage models, OA-Mf Interleukin 1 (IL1), chemokine (CXCL) and cytokine (CCL) family member profiles resemble the tolerized macrophage model, with the exception that OA-Mf show high levels of CCL20. CONCLUSION: Terminal osteoarthritis joints therefore harbor macrophages with an inflammatory state that closely resembles the tolerized macrophage state and this is compounded by a weak corticosteroid response capacity that may explain the lack of positive long-term effects of corticosteroid treatment for osteoarthritis patients.

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.

Epigenetic memory: A macrophage perspective.

The molecular basis of cellular memory is a fascinating topic that progressed with great strides during the last few decades. In the case of cells of the immune system, cellular memory likely extends beyond cell fate determination mechanisms, since immunity can tailor its responses to a potentially hostile environment that is a priori variable if not unpredictable. One particularly versatile innate immune system cell type is the macrophage. These phagocytes occur in all organs and tissues as resident cells or as differentiation products of recruited circulating blood monocytes. They come in many flavours determined by the tissue of residence and by external factors such as microbes. Recently, macrophage epigenome profiling has revealed thousands of chromosomal loci that are differentially active in macrophages, revealing chromosome elements that drive macrophage gene expression. The most dynamic epigenomic mark is nucleosomal histone acetylation. This mark is found at gene promoters and enhancers and correlates very well with gene expression changes. A second mark is H3K4me3, which sharply decorates the promoters of most protein coding genes that are (potentially) expressed. H3K4me3 at promoters is surrounded by its precursor H3K4me1. However, most often H3K4me1 occurs without H3K4me3 at enhancers where it appears together with histone acetylation, but can persist long after acetylation decreased. Hence, the biochemical signal H3K4me1 embodies appears to be a key to the plasticity of macrophage gene expression potential.

Cellular reprogramming for clinical cartilage repair.

The repair of articular cartilage needs a sufficient number of chondrocytes to replace the defect tissue, and therefore, expansion of cells is generally required. Chondrocytes derived by cellular reprogramming may provide a solution to the limitations of current (stem) cell-based therapies. In this article, two distinct approaches-induced pluripotent stem cell (iPSC)-mediated reprogramming and direct lineage conversion-are analysed and compared according to criteria that encompass the qualification of the method and the derived chondrocytes for the purpose of clinical application. Progress in iPSC generation has provided insights into the replacement of reprogramming factors by small molecules and chemical compounds. As follows, multistage chondrogenic differentiation methods have shown to improve the chondrocyte yield and quality. Nevertheless, the iPSC 'detour' remains a time- and cost-consuming approach. Direct conversion of fibroblasts into chondrocytes provides a slight advantage over these aspects compared to the iPSC detour. However, the requirement of constitutive transgene expression to inhibit hypertrophic differentiation limits this approach of being translated to the clinic. It can be concluded that the quality of the derived chondrocytes highly depends on the characteristics of the reprogramming method and that this is important to keep in mind during the experimental set-up. Further research into both reprogramming approaches for clinical cartilage repair has to include proper control groups and epigenetic profiling to optimize the techniques and eventually derive functionally stable articular chondrocytes.

Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy.

Nucleosome positioning dictates eukaryotic DNA compaction and access. To predict nucleosome positions in a statistical mechanics model, we exploited the knowledge that nucleosomes favor DNA sequences with specific periodically occurring dinucleotides. Our model is the first to capture both dyad position within a few base pairs, and free binding energy within 2 k(B)T, for all the known nucleosome positioning sequences. By applying Percus's equation to the derived energy landscape, we isolate sequence effects on genome-wide nucleosome occupancy from other factors that may influence nucleosome positioning. For both in vitro and in vivo systems, three parameters suffice to predict nucleosome occupancy with correlation coefficients of respectively 0.74 and 0.66. As predicted, we find the largest deviations in vivo around transcription start sites. This relatively simple algorithm can be used to guide future studies on the influence of DNA sequence on chromatin organization.

Chromatin accessibility, p300, and histone acetylation define PML-RARα and AML1-ETO binding sites in acute myeloid leukemia.

Chromatin accessibility plays a key role in regulating cell type specific gene expression during hematopoiesis but has also been suggested to be aberrantly regulated during leukemogenesis. To understand the leukemogenic chromatin signature, we analyzed acute promyelocytic leukemia, a subtype of leukemia characterized by the expression of RARα-fusion proteins, such as PML-RARα. We used nuclease accessibility sequencing in cell lines as well as patient blasts to identify accessible DNA elements and identified > 100 000 accessible regions in each case. Using ChIP-seq, we identified H2A.Z as a histone modification generally associated with these accessible regions, whereas unsupervised clustering analysis of other chromatin features, including DNA methylation, H2A.Zac, H3ac, H3K9me3, H3K27me3, and the regulatory factor p300, distinguished 6 distinct clusters of accessible sites, each with a characteristic functional makeup. Of these, PML-RARα binding was found specifically at accessible chromatin regions characterized by p300 binding and hypoacetylated histones. Identifying regions with a similar epigenetic make up in t(8;21) acute myeloid leukemia (AML) cells, another subtype of AMLs, revealed that these regions are occupied by the oncofusion protein AML1-ETO. Together, our results suggest that oncofusion proteins localize to accessible regions and that chromatin accessibility together with p300 binding and histone acetylation characterize AML1-ETO and PML-RARα binding sites.

Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes.

For efficient transcription, RNA PolII must overcome the presence of nucleosomes. The p38-related MAPK Hog1 is an important regulator of transcription upon osmostress in yeast and thereby it is involved in initiation and elongation. However, the role of this protein kinase in elongation has remained unclear. Here, we show that during stress there is a dramatic change in the nucleosome organization of stress-responsive loci that depends on Hog1 and the RSC chromatin remodelling complex. Upon stress, the MAPK Hog1 physically interacts with RSC to direct its association with the ORF of osmo-responsive genes. In RSC mutants, PolII accumulates on stress promoters but not in coding regions. RSC mutants also display reduced stress gene expression and enhanced sensitivity to osmostress. Cell survival under acute osmostress might thus depend on a burst of transcription that in turn could occur only with efficient nucleosome eviction. Our results suggest that the selective targeting of the RSC complex by Hog1 provides the necessary mechanistic basis for this event.

Corticosteroid-induced chromatin loop dynamics at the FKBP5 gene.

FKBP5 is a 115-kb-long glucocorticoid-inducible gene implicated in psychiatric disorders. To investigate the complexities of chromatin interaction frequencies at the FKBP5 topologically associated domain (TAD), we deployed 15 one-to-all chromatin capture viewpoints near gene promoters, enhancers, introns, and CTCF-loop anchors. This revealed a "one-TAD-one-gene" structure encompassing the FKBP5 promoter and its enhancers. The FKBP5 promoter and its two glucocorticoid-stimulated enhancers roam the entire TAD while displaying subtle cell type-specific interactomes. The FKBP5 TAD consists of two nested CTCF loops that are coordinated by one CTCF site in the eighth intron of FKBP5 and another beyond its polyadenylation site, 61 kb further. Loop extension correlates with transcription increases through the intronic CTCF site. This is efficiently compensated for, since the short loop is restored even under high transcription regimes. The boundaries of the FKBP5 TAD consist of divergent CTCF site patterns, harbor multiple smaller genes, and are resilient to glucocorticoid stimulation. Interestingly, both FKBP5 TAD boundaries harbor H3K27me3-marked heterochromatin blocks that may reinforce them. We propose that cis-acting genetic and epigenetic polymorphisms underlying FKBP5 expression variation are likely to reside within a 240-kb region that consists of the FKBP5 TAD, its left sub-TAD, and both its boundaries.

Quantification of absolute transcription factor binding affinities in the native chromatin context using BANC-seq.

Transcription factor binding across the genome is regulated by DNA sequence and chromatin features. However, it is not yet possible to quantify the impact of chromatin context on transcription factor binding affinities. Here, we report a method called binding affinities to native chromatin by sequencing (BANC-seq) to determine absolute apparent binding affinities of transcription factors to native DNA across the genome. In BANC-seq, a concentration range of a tagged transcription factor is added to isolated nuclei. Concentration-dependent binding is then measured per sample to quantify apparent binding affinities across the genome. BANC-seq adds a quantitative dimension to transcription factor biology, which enables stratification of genomic targets based on transcription factor concentration and prediction of transcription factor binding sites under non-physiological conditions, such as disease-associated overexpression of (onco)genes. Notably, whereas consensus DNA binding motifs for transcription factors are important to establish high-affinity binding sites, these motifs are not always strictly required to generate nanomolar-affinity interactions in the genome.

The human histone H3 complement anno 2011.

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.

The gene regulation knowledge commons: the action area of GREEKC.

As computational modeling becomes more essential to analyze and understand biological regulatory mechanisms, governance of the many databases and knowledge bases that support this domain is crucial to guarantee reliability and interoperability of resources. To address this, the COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC, CA15205, www.greekc.org) organized nine workshops in a four-year period, starting September 2016. The workshops brought together a wide range of experts from all over the world working on various steps in the knowledge management process that focuses on understanding gene regulatory mechanisms. The discussions between ontologists, curators, text miners, biologists, bioinformaticians, philosophers and computational scientists spawned a host of activities aimed to standardize and update existing knowledge management workflows and involve end-users in the process of designing the Gene Regulation Knowledge Commons (GRKC). Here the GREEKC consortium describes its main achievements in improving this GRKC.

Genetic identification of a network of factors that functionally interact with the nucleosome remodeling ATPase ISWI.

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

Fibronectin-functionalization of 3D collagen networks supports immune tolerance and inflammation suppression in human monocyte-derived macrophages.

The extracellular matrix (ECM) is dynamically reorganized during wound healing. Concomitantly, recruited monocytes differentiate into macrophages. However, the role of the wound's ECM during this transition remain to be fully understood. Fibronectin is a multifunctional glycoprotein present in early wound ECM with a potential immunomodulatory role during monocyte-to-macrophage differentiation. Hence, to investigate the impact of fibronectin during this differentiation step, 3D fibrillar collagen type I networks with or without fibronectin-functionalization were engineered with defined topology (fibril and pore diameter: 0.8 µm; 7 µm) and amount of adsorbed fibronectin (0.15 µg per µg collagen). Primary, human monocytes were then differentiated into macrophages inside these networks. The immunological imprinting of the resulting macrophages was monitored by means of the expression of FABP4, CLEC4E, SLC2A6, and SOD2 which discriminate naïve and tolerized macrophages, as well pro-inflammatory (M1) and anti-inflammatory (M2) macrophage polarization. The analyses indicate that fibronectin-functionalization of collagen I networks induces macrophage tolerance rather than M1 or M2 macrophage phenotypes. This finding was confirmed by release profiles of pro- and anti-inflammatory cytokines such as IL6, IL8, CXCL10, and IL10. Nevertheless, upon LPS challenge, immune suppression by fibronectin was overridden since these macrophages could then deploy an efficient immune response. Our results therefore provide new perspectives in biomaterial science of wound healing scaffolds and the design of instructive materials for human monocyte-derived cells.

Gene Ontology representation for transcription factor functions.

Transcription plays a central role in defining the identity and functionalities of cells, as well as in their responses to changes in the cellular environment. The Gene Ontology (GO) provides a rigorously defined set of concepts that describe the functions of gene products. A GO annotation is a statement about the function of a particular gene product, represented as an association between a gene product and the biological concept a GO term defines. Critically, each GO annotation is based on traceable scientific evidence. Here, we describe the different GO terms that are associated with proteins involved in transcription and its regulation, focusing on the standard of evidence required to support these associations. This article is intended to help users of GO annotations understand how to interpret the annotations and can contribute to the consistency of GO annotations. We distinguish between three classes of activities involved in transcription or directly regulating it - general transcription factors, DNA-binding transcription factors, and transcription co-regulators.

Integration of transcription coregulator complexes with sequence-specific DNA-binding factor interactomes.

The domain of transcription regulation has been notoriously difficult to annotate in the Gene Ontology, partly because of the intricacies of gene regulation which involve molecular interactions with DNA as well as amongst protein complexes. The molecular function 'transcription coregulator activity' is a part of the biological process 'regulation of transcription, DNA-templated' that occurs in the cellular component 'chromatin'. It can mechanistically link sequence-specific DNA-binding transcription factor (dbTF) regulatory DNA target sites to coactivator and corepressor target sites through the molecular function 'cis-regulatory region sequence-specific DNA binding'. Many questions arise about transcription coregulators (coTF). Here, we asked how many unannotated, putative coregulators can be identified in protein complexes? Therefore, we mined the CORUM and hu.MAP protein complex databases with known and strongly presumed human transcription coregulators. In addition, we trawled the BioGRID and IntAct molecular interaction databases for interactors of the known 1457 human dbTFs annotated by the GREEKC and GO consortia. This yielded 1093 putative transcription factor coregulator complex subunits, of which 954 interact directly with a dbTF. This substantially expands the set of coTFs that could be annotated to 'transcription coregulator activity' and sets the stage for renewed annotation and wet-lab research efforts. To this end, we devised a prioritisation score based on existing GO annotations of already curated transcription coregulators as well as interactome representation. Since all the proteins that we mined are parts of protein complexes, we propose to concomitantly engage in annotation of the putative transcription coregulator-containing complexes in the Complex Portal database.

Sequence Ontology terminology for gene regulation.

The Sequence Ontology (SO) is a structured, controlled vocabulary that provides terms and definitions for genomic annotation. The Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC) initiative has gathered input from many groups of researchers, including the SO, the Gene Ontology (GO), and gene regulation experts, with the goal of curating information about how gene expression is regulated at the molecular level. Here we discuss recent updates to the SO reflecting current knowledge. We have developed more accurate human-readable terms (also known as classes), including new definitions, and relationships related to the expression of genes. New findings continue to give us insight into the biology of gene regulation, including the order of events, and participants in those events. These updates to the SO support logical reasoning with the current understanding of gene expression regulation at the molecular level.

A GO catalogue of human DNA-binding transcription factors.

To control gene transcription, DNA-binding transcription factors recognise specific sequence motifs in gene regulatory regions. A complete and reliable GO annotation of all DNA-binding transcription factors is key to investigating the delicate balance of gene regulation in response to environmental and developmental stimuli. The need for such information is demonstrated by the many lists of transcription factors that have been produced over the past decade. The COST Action Gene Regulation Ensemble Effort for the Knowledge Commons (GREEKC) Consortium brought together experts in the field of transcription with the aim of providing high quality and interoperable gene regulatory data. The Gene Ontology (GO) Consortium provides strict definitions for gene product function, including factors that regulate transcription. The collaboration between the GREEKC and GO Consortia has enabled the application of those definitions to produce a new curated catalogue of over 1400 human DNA-binding transcription factors, that can be accessed at https://www.ebi.ac.uk/QuickGO/targetset/dbTF. This catalogue has facilitated an improvement in the GO annotation of human DNA-binding transcription factors and led to the GO annotation of almost sixty thousand DNA-binding transcription factors in over a hundred species. Thus, this work will aid researchers investigating the regulation of transcription in both biomedical and basic science.

Formalization of gene regulation knowledge using ontologies and gene ontology causal activity models.

Gene regulation computational research requires handling and integrating large amounts of heterogeneous data. The Gene Ontology has demonstrated that ontologies play a fundamental role in biological data interoperability and integration. Ontologies help to express data and knowledge in a machine processable way, which enables complex querying and advanced exploitation of distributed data. Contributing to improve data interoperability in gene regulation is a major objective of the GREEKC Consortium, which aims to develop a standardized gene regulation knowledge commons. GREEKC proposes the use of ontologies and semantic tools for developing interoperable gene regulation knowledge models, which should support data annotation. In this work, we study how such knowledge models can be generated from cartoons of gene regulation scenarios. The proposed method consists of generating descriptions in natural language of the cartoons; extracting the entities from the texts; finding those entities in existing ontologies to reuse as much content as possible, especially from well known and maintained ontologies such as the Gene Ontology, the Sequence Ontology, the Relations Ontology and ChEBI; and implementation of the knowledge models. The models have been implemented using Protégé, a general ontology editor, and Noctua, the tool developed by the Gene Ontology Consortium for the development of causal activity models to capture more comprehensive annotations of genes and link their activities in a causal framework for Gene Ontology Annotations. We applied the method to two gene regulation scenarios and illustrate how to apply the models generated to support the annotation of data from research articles.