Epigenetic control of the genome exists at multiple levels, including nucleosomal binding to DNA, covalent modifications of DNA-associated proteins such as nucleosome histones, direct chemical modification of DNA (such as DNA methylation, hydroxymethylation) and high-order chromatin structure and phase condensation41,42. The epigenome, also termed the ‘epigenomic landscape’ is the genome-wide pattern of chromatin accessibility, DNA modification, and binding of transcription factors and chromatin-modifying enzymes, and is unique to each cell type. The epigenome controls gene expression through various mechanisms, including regulating transcription factor access, and organizing the nuclear architecture of the chromosomes. Furthermore, during inflammatory processes or pathogenic conditions, epigenetic patterns are plastic, and epigenetic regulation has been implicated in inflammatory responses and disease states43-45. Given the importance of the epigenome in defining cell identity and ensuring normal cellular function, a better understanding of how the abnormal changes in the epigenetic profile lead to cellular dysregulation and rheumatic diseases is needed. Insights into cellular epigenetic landscapes can be achieved from high-throughput, genome-wide methods for analyzing chromatin accessibility, histone modification, transcription factor occupancy and DNA methylation45,46. These include DNase-seq, FAIRE-seq, and ATAC-seq, which reveal nucleosome-depleted regions known as open chromatin. ChIP-seq using specific antibodies for histone marks or transcription factors reveals genome-wide patterns of histone modifications, which can promote, repress or stabilize gene expression, and transcription factor occupancy (DNA methylation will be discussed in the section below).
ChIP-seq, ATACseq, HiChIP.
Genome-wide epigenetic analysis for histone modifications and chromatin accessibility in rheumatic diseases is still at an early stage, although epigenetic changes at disease-relevant genes have been detected in rheumatic disease samples47. Gene enhancers are altered in monocytes in systemic lupus erythematosus (SLE)48. Both promoters and enhancers exhibit significant changes in histone methylation in SLE. Regions with differential histone 3 lysine 4 trimethylation (H3K4me3) in SLE are significantly enriched in potential interferon-related transcription factor binding sites and pioneer transcription factor sites49. An SLE specific chromatin accessibility signature of B cells was also identified using ATAC-seq on biobanked specimens50. Changes in accessibility occur at loci surrounding genes involved in B cell activation. In RA fibroblast-like synoviocytes (FLS)51, epigenetic profiling of various histone modifications such as H3K27ac, H3K4me1, H3K4me3 and H3K36me3 and open chromatin has been performed. Epigenomically similar regions exist in RA FLS cells relative to OA controls and are associated with active enhancers and promoters of immune-related genes. Although these studies represent an important step forward for epigenomic analysis in complex rheumatic diseases, it is still a major challenge to translate epigenetic regulation into patterns of gene expression that define cell activation state and phenotype.
Another important concept that will be useful in future research is that chromatin-mediated epigenetic mechanisms participate in memory-like phenomena that can either enhance or inhibit inflammatory responses52, which may provide new insights to interpret the dysregulation of immune response in rheumatic diseases. Epigenetically mediated immunological imprinting can manifest as tolerance53 or training54, which suppress inflammation or enhance it, respectively. Tolerant macrophages exhibit a selective downregulation of chromatin accessibility and active histone marks such as H3K4me3 and H3K27ac at promoters and enhancers of inflammatory genes55,56, as well as a defect in TLR signaling53. In trained monocytes, distal regulatory elements gain H3K27ac and persistent H3K4me1 marks. Upon re-stimulation, genomic regions containing H3K4me1 marks are quickly recognized and acetylated to promote rapid and enhanced transcriptional responses54. Both tolerance and training represent clinically relevant functional states and the development of innate memory has been shown to be relevant to inflammation52,53.
Tolerance is part of the immune paralysis that occurs during bacterial sepsis or late phases of septic shock. Interestingly, exposure to different LPS subtypes produced by the gut microbiome can either stimulate or inhibit endotoxin tolerance and alter the course of autoimmunity such as the incidence of diabetes57. These observations suggest that microbiome-derived LPS could impact long-term immunosuppressive mechanisms in more complex ways. Also, type I interferon, which is an abundant cytokine in many rheumatic diseases, can abolish macrophage tolerance in a chromatin-dependent manner55,58. Conversely to tolerance mechanisms, epigenetic-mediated training of monocytes can contribute to nonspecific protective effects of vaccination that strongly influence susceptibility to secondary infections52. The induction of trained immunity by ?-glucan is able to counteract the epigenetic changes induced in monocytes in post-sepsis immunoparalysis56. A recent study suggested that peripherally applied inflammatory stimuli induce acute immune training and tolerance in the brain and lead to differential epigenetic reprogramming of microglia that persists for at least six months59. This study supports the concept that environmental stimuli acting at distal sites can affect inflammatory responses in distinct tissues such as RA synovium, thereby contributing to pathogenesis. Overall, tolerance and training are likely initial examples of a more pervasive phenomenon of epigenetic-mediated conditioning by exposure to environmental challenges including chronic inflammation. Induction of innate immune memory may be important for diseases characterized by dysregulation of immune responses and it will be interesting to determine whether defects in establishing tolerance and training states contribute to rheumatic diseases.
Numerous studies have shown that the vast majority of disease-associated allelic variants fall outside protein-coding sequences and instead lie in cis-regulatory regions such as gene enhancers60-64; this includes common autoimmune-disease associated polymophisms65. Lupus associated polymorphisms in the HLA and BANK1 loci have been studied in depth through large-scale efforts, and have been implicated as eQTLs that control gene expression66,67. As few disease-associated eQTLs have been mapped relative to large numbers of disease-associated SNVs, it is a major challenge to assign distal enhancers that harbor disease-associated allelic variants to their target genes, and to determine causal relationships between these variants, gene expression and disease states. A major advance that begins to address the causal link between chromatin landscape and function (gene expression) came with the development of tools to study chromatin loop (re)organization42 and CRISPR-Cas9 genome editing68. Mapping of chromatin contacts has tremendously advanced in the last decade owing to the development of chromosomal conformation capture techniques such as Hi-C42. However, because Hi-C interrogates all possible interactions genome wide, deep sequencing is required to fully identify chromatin architectural features. Interestingly, a recent study used the histone modification that correlates with active enhancers and promoters (H3K27ac) as a bait for their recently developed HiChIP method, a combination of ChIP and Hi-C assays for mapping protein-centric chromatin interactions in as few as 50,000 cells69. They generated high-resolution maps of enhancer–promoter contacts in primary human naive CD4+ T cells, Treg cells and Th17 cells. When overlapping autoimmune disease–associated variants in intergenic regions with the interaction loops, they found that enhancers harboring disease-associated variants interacted with between zero to ten target genes, thereby identifying target genes regulated by potentially causal SNVs. This approach will provide a valuable framework for mapping distal cis-regulatory regions and disease-associated variants to target genes in rheumatic diseases.
For the functional study of epigenetic regulation, recently CRISPR-Cas9-mediated genome editing has provided the opportunity to manipulate the epigenome and observe the effects that it may have on cell function, development, differentiation and activation68. This approach has been used to support the disease-relevant importance of an enhancer in the TNFAIP3 locus70. Although most studies center on the ability to insert or remove genes or to repair disease-causing mutations, CRISPR-Cas9 has been further expanded with the engineering of a nuclease-deficient version, dCas9, which can be used to directly manipulate a specific regulatory region or epigenetic marks to determine the impact on transcriptional activity and cellular functions. The dCas9-based tool can be fused with a known functional domain from proteins such as KRAB, LSD1, TET1 or DNMT3A, in order to repress gene transcription. On the other hand, dCas9 may be used to induce gene expression through the activation domains of several factors such as p300 or VP64. Another CRISPR-Cas9 technique using modified guide RNA, called target gene activation, was used in mice to treat several different diseases such as diabetes, muscular dystrophy, and acute kidney disease71. Overall, the CRISPR-Cas9 system can be used to determine enhancer regions that are associated with a gene of interest and will help to uncover pressing epigenetic questions involving the precise role of each histone modifying enzyme, the interaction between cis-regulatory regions and target genes, and the effect of histone modifications on gene regulation in rheumatic diseases. Manipulating aberrant, disease-causing epigenetic marks would thus seem to hold considerable therapeutic promise.