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Since Conrad Waddington has introduced a conceptual definition of the epigenetic landscape to describe the interaction of genes with their environment1, it is now clear that epigenetic regulation is crucially important for various other biological processes such as development, differentiation, proliferation and activation2,3. Epigenetic regulation is mediated in various layers of the chromatin structure, including DNA packaging around nucleosomes, covalent modifications of histone tails, DNA methylation and high-order chromatin folding3,4.
The epigenome can differ from cell type to cell type and can potentially regulate gene expression in many ways by inhibiting or promoting transcription factor access to DNA, mediating gene expression, and organizing the nuclear architecture of the chromosomes. Furthermore, during inflammatory processes or pathogenic conditions, epigenetic patterns are plastic, and regulated gene expression plays an important role in inflammatory processes and disease states5-7. Given the importance of the epigenome to define cell identity and to ensure normal cellular function, a better understanding of how the abnormal changes in the epigenetic profile lead to cellular dysregulation and diseases is needed.
Over the past decade, epigenetic profiling has been important in discovering associations between epigenetic alterations and disease. The main insight into epigenetic landscape has come from high-throughput, genome-wide methods for analyzing chromatin accessibility, histone modification, transcription factor occupancy and DNA methylation7,8. 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 marks and transcription factor occupancy (DNA methylation will be discussed in the section below). This genome-wide sequencing technology can be used for non-biased profiling of open chromatin, chromatin remodelers, transcription factors, and other proteins that are associated with DNA as well as histone modifications. Genome-wide epigenetic analysis for histone modifications and chromatin accessibility in rheumatic diseases is still lacking, although epigenetic changes at disease-relevant genes have been shown to play an important role in many rheumatic diseases9. It has been shown that monocyte enhancers are highly altered in systemic lupus erythematosus (SLE)10. Both promoters and enhancers exhibited significant changes in histone methylation in SLE. Regions with differential H3K4me3 in SLE were significantly enriched in potential interferon-related transcription factor binding sites and pioneer transcription factor sites11. An SLE specific chromatin accessibility signature of B cells was also identified using ATAC-seq on biobanked specimens12. Changes in accessibility have occurred at loci surrounding genes involved in B cell activation. In RA fibroblast-like synoviocytes (FLS)13, epigenetic profiling of various histone modifications such as H3K27ac, H3K4me1, H3K4me3 and H3K36me3 and open chromatin has been shown. Epigenomically similar regions exist in RA FLS cells and are associated with active enhancers and promoters of immune-related genes. Although these studies represent an important step forward for mapping epigenetic landscape in complex rheumatic diseases, it is still a major challenge to translate the epigenetic regulation into molecular mechanisms.
Another important concept that will be useful in future research is that chromatin-mediated epigenetic mechanisms also participate in memory-like phenomena that can either enhance or inhibit inflammatory response14, which may provide new insights to interpret the dysregulation of immune response in rheumatic diseases. Epigenetically mediated immunological imprinting can manifest as tolerance15 or training16, which suppress inflammation or enhance it, respectively. Tolerant macrophages exhibit a selective downregulation for chromatin accessibility and active histone marks such as H3K4me3 and H3K27ac at promoters and enhancers of inflammatory genes17,18, as well as a defect in TLR signaling15. In trained monocytes, distal regulatory elements gained H3K27ac and persistant H3K4me1 marks. Upon re-stimulus, genomic regions containing H3K4me1 marks are quickly recognized and assigned to regain H3K27ac marks for rapid and enhanced transcriptional response16. Both tolerance and training represent clinically relevant functional states and the development of innate memory has been shown to be relevant to inflammation14,15. Tolerance could induce the immune paralysis encountered during bacterial sepsis or endotoxic shock. Interestingly, exposure to different LPS subtypes produced by the gut microbiome could either stimulate or inhibit endotoxin tolerance and alter the course of autoimmunity such as the incidence of diabetes19. 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 cytokines in many rheumatic diseases, can abolish macrophage tolerance in a chromatin-dependent manner17,20. Similar to tolerance mechanisms, epigenetic-mediated training of monocytes can provide the nonspecific protective effects of live microorganism vaccination that strongly influence susceptibility to secondary infections14. The induction of trained immunity by ?-glucan is able to counteract the epigenetic changes induced in monocytes in post-sepsis immunoparalysis18. 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 months21. Overall, tolerance and training are likely initial examples of a more pervasive phenomenon of epigenetic-mediated conditioning by exposure to environmental changes including chronic inflammation. Induction of innate immune memory may be important for diseases characterized by defective function 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 variants or cis-regulatory regions such as enhancers fall outside of protein-coding sequence22-24. Thus, it has been a major challenge in this field how to assign disease variants or distal enhancers to their target genes and how to determine a cause and effect relationship between a distinct epigenetic mark and the function of the affected genes. A major advance that begins to address the causal link with chromatin landscape and function came with the development of chromatin loop reorganization4 and CRISPR-Cas9 tools25. Mapping of chromatin contacts has tremendously advanced in the last decade owing to the development of chromosomal conformation capture techniques such as Hi-C4. However, because Hi-C interrogates all possible proximity ligations 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 cells26. They generated high-resolution maps of enhancer–promoter contacts in primary 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 the disease-associated variants interacted with from zero to ten target genes. This study 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 regulations, recent CRISPR-Cas9 has provided the opportunity to manipulate the epigenome and observe the effects that it may have on cell function, development, differentiation and activation25. Although most studies center on the ability to insert or remove genes or to repair disease-causing mutations, the CRISPR-Cas9 has been further expanded with the engineering of the 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, 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 the modified guide RNA, called target gene activation, was used in mice to treat several different diseases such as diabetes, muscular dystrophy, and acute kidney disease27. 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, the effect of these histone modifications and gene regulation in rheumatic diseases. Manipulating aberrant, disease-causing epigenetic marks would thus seem to hold considerable therapeutic promise.

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