Mapping the epigenomic landscape of human monocytes following innate immune activation reveals context-specific mechanisms driving endotoxin tolerance.

BACKGROUND: Monocytes are key mediators of innate immunity to infection, undergoing profound and dynamic changes in epigenetic state and immune function which are broadly protective but may be dysregulated in disease. Here, we aimed to advance understanding of epigenetic regulation following innate immune activation, acutely and in endotoxin tolerant states. METHODS: We exposed human primary monocytes from healthy donors (n = 6) to interferon-γ or differing combinations of endotoxin (lipopolysaccharide), including acute response (2 h) and two models of endotoxin tolerance: repeated stimulations (6 + 6 h) and prolonged exposure to endotoxin (24 h). Another subset of monocytes was left untreated (naïve). We identified context-specific regulatory elements based on epigenetic signatures for chromatin accessibility (ATAC-seq) and regulatory non-coding RNAs from total RNA sequencing. RESULTS: We present an atlas of differential gene expression for endotoxin and interferon response, identifying widespread context specific changes. Across assayed states, only 24-29% of genes showing differential exon usage are also differential at the gene level. Overall, 19.9% (6,884 of 34,616) of repeatedly observed ATAC peaks were differential in at least one condition, the majority upregulated on stimulation and located in distal regions (64.1% vs 45.9% of non-differential peaks) within which sequences were less conserved than non-differential peaks. We identified enhancer-derived RNA signatures specific to different monocyte states that correlated with chromatin accessibility changes. The endotoxin tolerance models showed distinct chromatin accessibility and transcriptomic signatures, with integrated analysis identifying genes and pathways involved in the inflammatory response, detoxification, metabolism and wound healing. We leveraged eQTL mapping for the same monocyte activation states to link potential enhancers with specific genes, identifying 1,946 unique differential ATAC peaks with 1,340 expression associated genes. We further use this to inform understanding of reported GWAS, for example involving FCHO1 and coronary artery disease. CONCLUSION: This study reports context-specific regulatory elements based on transcriptomic profiling and epigenetic signatures for enhancer-derived RNAs and chromatin accessibility in immune tolerant monocyte states, and demonstrates the informativeness of linking such elements and eQTL to inform future mechanistic studies aimed at defining therapeutic targets of immunosuppression and diseases.

Comprehensive epigenomic profiling reveals the extent of disease-specific chromatin states and informs target discovery in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a common, highly heritable inflammatory arthritis characterized by enthesitis of the spine and sacroiliac joints. Genome-wide association studies (GWASs) have revealed more than 100 genetic associations whose functional effects remain largely unresolved. Here, we present a comprehensive transcriptomic and epigenomic map of disease-relevant blood immune cell subsets from AS patients and healthy controls. We find that, while CD14+ monocytes and CD4+ and CD8+ T cells show disease-specific differences at the RNA level, epigenomic differences are only apparent upon multi-omics integration. The latter reveals enrichment at disease-associated loci in monocytes. We link putative functional SNPs to genes using high-resolution Capture-C at 10 loci, including PTGER4 and ETS1, and show how disease-specific functional genomic data can be integrated with GWASs to enhance therapeutic target discovery. This study combines epigenetic and transcriptional analysis with GWASs to identify disease-relevant cell types and gene regulation of likely pathogenic relevance and prioritize drug targets.

Epigenomic analysis reveals a dynamic and context-specific macrophage enhancer landscape associated with innate immune activation and tolerance.

BACKGROUND: Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications. RESULTS: We describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA. CONCLUSIONS: Our study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.

Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template.

Next generation sequencing methods that can be applied to both the resequencing of whole genomes and to the selective resequencing of specific parts of genomes are needed. We describe (i) a massively scalable biochemistry, Cyclical Ligation and Cleavage (CycLiC) for contiguous base sequencing and (ii) apply it directly to a template captured on a microarray. CycLiC uses four color-coded DNA/RNA chimeric oligonucleotide libraries (OL) to extend a primer, a base at a time, along a template. The cycles comprise the steps: (i) ligation of OLs, (ii) identification of extended base by label detection, and (iii) cleavage to remove label/terminator and undetermined bases. For proof-of-principle, we show that the method conforms to design and that we can read contiguous bases of sequence correctly from a template captured by hybridization from solution to a microarray probe. The method is amenable to massive scale-up, miniaturization and automation. Implementation on a microarray format offers the potential for both selection and sequencing of a large number of genomic regions on a single platform. Because the method uses commonly available reagents it can be developed further by a community of users.

Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms.

Association studies using common sequence variants or single nucleotide polymorphisms (SNPs) may provide a powerful approach to dissect the genetic inheritance of common complex traits. Such studies necessitate the development of cost-effective, high throughput technologies for scoring SNPs. The method described in this paper for the co-detection of both alleles of a SNP in a single homogeneous reaction combines the specificity of a high fidelity DNA ligation step with the power of rolling circle amplification. The incorporation of Amplifluor energy transfer primers enables signal detection in a homogeneous format, making this approach highly amenable to automation. The adaptation of the genotyping method for high throughput screening using conventional liquid handling systems is described.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors.

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Optimisation of a triazolopyridine based histone demethylase inhibitor yields a potent and selective KDM2A (FBXL11) inhibitor.

A potent inhibitor of the JmjC histone lysine demethylase KDM2A (compound 35, pIC50 7.2) with excellent selectivity over representatives from other KDM subfamilies has been developed; the discovery that a triazolopyridine compound binds to the active site of JmjC KDMs was followed by optimisation of the triazole substituent for KDM2A inhibition and selectivity.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.