Functional Hallmarks of Healthy Macrophage Responses: Their Regulatory Basis and Disease Relevance.

Macrophages are first responders for the immune system. In this role, they have both effector functions for neutralizing pathogens and sentinel functions for alerting other immune cells of diverse pathologic threats, thereby initiating and coordinating a multipronged immune response. Macrophages are distributed throughout the body-they circulate in the blood, line the mucosal membranes, reside within organs, and survey the connective tissue. Several reviews have summarized their diverse roles in different physiological scenarios and in the initiation or amplification of different pathologies. In this review, we propose that both the effector and the sentinel functions of healthy macrophages rely on three hallmark properties: response specificity, context dependence, and stimulus memory. When these hallmark properties are diminished, the macrophage's biological functions are impaired, which in turn results in increased risk for immune dysregulation, manifested by immune deficiency or autoimmunity. We review the evidence and the molecular mechanisms supporting these functional hallmarks.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells.

The long non-coding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here, we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-type-specific XIST complexes and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. Single-cell transcriptome data of female patients with either systemic lupus erythematosus or COVID-19 infection revealed XIST dysregulation, reflected by escape of XIST-dependent genes, in CD11c+ atypical memory B cells (ABCs). XIST inactivation with TLR7 agonism suffices to promote isotype-switched ABCs. These results indicate cell-type-specific diversification and function for lncRNA-protein complexes and suggest expanded roles for XIST in sex-differences in biology and medicine.

Evolutionary Persistence of DNA Methylation for Millions of Years after Ancient Loss of a De Novo Methyltransferase.

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules.

Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating "read-write" architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells.

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.

Asymmetric Histone Inheritance: Establishment, Recognition, and Execution.

The discovery of biased histone inheritance in asymmetrically dividing Drosophila melanogaster male germline stem cells demonstrates one means to produce two distinct daughter cells with identical genetic material. This inspired further studies in different systems, which revealed that this phenomenon may be a widespread mechanism to introduce cellular diversity. While the extent of asymmetric histone inheritance could vary among systems, this phenomenon is proposed to occur in three steps: first, establishment of histone asymmetry between sister chromatids during DNA replication; second, recognition of sister chromatids carrying asymmetric histone information during mitosis; and third, execution of this asymmetry in the resulting daughter cells. By compiling the current knowledge from diverse eukaryotic systems, this review comprehensively details and compares known chromatin factors, mitotic machinery components, and cell cycle regulators that may contribute to each of these three steps. Also discussed are potential mechanisms that introduce and regulate variable histone inheritance modes and how these different modes may contribute to cell fate decisions in multicellular organisms.

Replicational Dilution of H3K27me3 in Mammalian Cells and the Role of Poised Promoters.

Polycomb repressive complex 2 (PRC2) places H3K27me3 at developmental genes and is causally implicated in keeping bivalent genes silent. It is unclear if that silence requires minimum H3K27me3 levels and how the mark transmits faithfully across mammalian somatic cell generations. Mouse intestinal cells lacking EZH2 methyltransferase reduce H3K27me3 proportionately at all PRC2 target sites, but ∼40% uniform residual levels keep target genes inactive. These genes, derepressed in PRC2-null villus cells, remain silent in intestinal stem cells (ISCs). Quantitative chromatin immunoprecipitation and computational modeling indicate that because unmodified histones dilute H3K27me3 by 50% each time DNA replicates, PRC2-deficient ISCs initially retain sufficient H3K27me3 to avoid gene derepression. EZH2 mutant human lymphoma cells also require multiple divisions before H3K27me3 dilution relieves gene silencing. In both cell types, promoters with high basal H3K4me2/3 activate in spite of some residual H3K27me3, compared to less-poised promoters. These findings have implications for PRC2 inhibition in cancer therapy.

STAT1 is required to establish but not maintain interferon-γ-induced transcriptional memory.

Exposure of human cells to interferon-γ (IFNγ) results in a mitotically heritable yet reversible state called long-term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here, we discovered that in primed cells, both interferon-responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re-exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ-induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK-STAT signaling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that the memory of interferon exposure constitutes a STAT1-mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

Establishment and maintenance of random monoallelic expression.

This Review elucidates the regulatory principles of random monoallelic expression by focusing on two well-studied examples: the X-chromosome inactivation regulator Xist and the olfactory receptor gene family. Although the choice of a single X chromosome or olfactory receptor occurs in different developmental contexts, common gene regulatory principles guide monoallelic expression in both systems. In both cases, an event breaks the symmetry between genetically and epigenetically identical copies of the gene, leading to the expression of one single random allele, stabilized through negative feedback control. Although many regulatory steps that govern the establishment and maintenance of monoallelic expression have been identified, key pieces of the puzzle are still missing. We provide an overview of the current knowledge and models for the monoallelic expression of Xist and olfactory receptors. We discuss their similarities and differences, and highlight open questions and approaches that could guide the study of other monoallelically expressed genes.

Extensive Recovery of Embryonic Enhancer and Gene Memory Stored in Hypomethylated Enhancer DNA.

Developing and adult tissues use different cis-regulatory elements. Although DNA at some decommissioned embryonic enhancers is hypomethylated in adult cells, it is unknown whether this putative epigenetic memory is complete and recoverable. We find that, in adult mouse cells, hypomethylated CpG dinucleotides preserve a nearly complete archive of tissue-specific developmental enhancers. Sites that carry the active histone mark H3K4me1, and are therefore considered "primed," are mainly cis elements that act late in organogenesis. In contrast, sites decommissioned early in development retain hypomethylated DNA as a singular property. In adult intestinal and blood cells, sustained absence of polycomb repressive complex 2 indirectly reactivates most-and only-hypomethylated developmental enhancers. Embryonic and fetal transcriptional programs re-emerge as a result, in reverse chronology to cis element inactivation during development. Thus, hypomethylated DNA in adult cells preserves a "fossil record" of tissue-specific developmental enhancers, stably marking decommissioned sites and enabling recovery of this epigenetic memory.

Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations.

Small RNAs trigger the formation of epialleles that are silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. Here, we demonstrate that small interfering RNA-induced epimutations in fission yeast are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. This process is mediated by the deposition of a phenotypically neutral molecular mark composed of tri-methylated histone H3 lysine 9 (H3K9me3). Its stable propagation is coupled to RNAi and requires maximal binding affinity of the Clr4/Suvar39 chromodomain to H3K9me3. In wild-type cells, this mark has no visible impact on transcription but causes gene silencing if RNA polymerase-associated factor 1 complex (Paf1C) activity is impaired. In sum, our results reveal a distinct form of epigenetic memory in which cells acquire heritable, transcriptionally active epialleles that confer gene silencing upon modulation of Paf1C.

Widespread Enhancer Dememorization and Promoter Priming during Parental-to-Zygotic Transition.

The epigenome plays critical roles in controlling gene expression and development. However, how the parental epigenomes transit to the zygotic epigenome in early development remains elusive. Here we show that parental-to-zygotic transition in zebrafish involves extensive erasure of parental epigenetic memory, starting with methylating gametic enhancers. Surprisingly, this occurs even prior to fertilization for sperm. Both parental enhancers lose histone marks by the 4-cell stage, and zygotic enhancers are not activated until around zygotic genome activation (ZGA). By contrast, many promoters remain hypomethylated and, unexpectedly, acquire histone acetylation before ZGA at as early as the 4-cell stage. They then resolve into either activated or repressed promoters upon ZGA. Maternal depletion of histone acetyltransferases results in aberrant ZGA and early embryonic lethality. Finally, such reprogramming is largely driven by maternal factors, with zygotic products mainly contributing to embryonic enhancer activation. These data reveal widespread enhancer dememorization and promoter priming during parental-to-zygotic transition.

Emergence and properties of adult mammalian epidermal stem cells.

In this review we discuss how the mammalian interfollicular epidermis forms during development, maintains homeostasis, and is repaired following wounding. Recent studies have provided new insights into the relationship between the stem cell compartment and the differentiating cell layers; the ability of differentiated cells to dedifferentiate into stem cells; and the epigenetic memory of epidermal cells following wounding.

CHROMOMETHYLTRANSFERASE3/KRYPTONITE maintains the sulfurea paramutation in Solanum lycopersicum.

SignificanceParamutation involves the transfer of a repressive epigenetic mark between silent and active alleles. It is best known from exceptional non-Mendelian inheritance of conspicuous phenotypes in maize but also in other plants and animals. Recent genomic studies, however, indicate that paramutation may be less exceptional. It may be a consequence of wide-cross hybridization and may contribute to quantitative trait variation or unstable phenotypes in crops. Using the sulfurea (sulf) locus in tomato, we demonstrate that a self-reinforcing feedback loop involving DNA- and histone-methyl transferases CHROMOMETHYLTRANSFERASE3 (CMT3) and KRYPTONITE (KYP) is required for paramutation of sulf and that there is a change in chromatin organization. These findings advance the understanding of non-Mendelian inheritance in plants.

Learning of Signaling Networks: Molecular Mechanisms.

Molecular processes of neuronal learning have been well described. However, learning mechanisms of non-neuronal cells are not yet fully understood at the molecular level. Here, we discuss molecular mechanisms of cellular learning, including conformational memory of intrinsically disordered proteins (IDPs) and prions, signaling cascades, protein translocation, RNAs [miRNA and long noncoding RNA (lncRNA)], and chromatin memory. We hypothesize that these processes constitute the learning of signaling networks and correspond to a generalized Hebbian learning process of single, non-neuronal cells, and we discuss how cellular learning may open novel directions in drug design and inspire new artificial intelligence methods.

Epigenetic weapons of plants against fungal pathogens.

In the natural environment, plants face constant exposure to biotic stress caused by fungal attacks. The plant's response to various biotic stresses relies heavily on its ability to rapidly adjust the transcriptome. External signals are transmitted to the nucleus, leading to activation of transcription factors that subsequently enhance the expression of specific defense-related genes. Epigenetic mechanisms, including histone modifications and DNA methylation, which are closely linked to chromatin states, regulate gene expression associated with defense against biotic stress. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense against stressors. These molecular modifications enable plants to exhibit enhanced resistance and productivity under diverse environmental conditions. Epigenetic mechanisms also contribute to stress-induced environmental epigenetic memory and priming in plants, enabling them to recall past molecular experiences and utilize this stored information for adaptation to new conditions. In the arms race between fungi and plants, a significant aspect is the cross-kingdom RNAi mechanism, whereby sRNAs can traverse organismal boundaries. Fungi utilize sRNA as an effector molecule to silence plant resistance genes, while plants transport sRNA, primarily through extracellular vesicles, to pathogens in order to suppress virulence-related genes. In this review, we summarize contemporary knowledge on epigenetic mechanisms of plant defense against attack by pathogenic fungi. The role of epigenetic mechanisms during plant-fungus symbiotic interactions is also considered.

Epigenetic differences between wild and cultivated grapevines highlight the contribution of DNA methylation during crop domestication.

The domestication process in grapevines has facilitated the fixation of desired traits. Nowadays, vegetative propagation through cuttings enables easier preservation of these genotypes compared to sexual reproduction. Nonetheless, even with vegetative propagation, various phenotypes are often present within the same vineyard due to the accumulation of somatic mutations. These mutations are not the sole factors influencing phenotype. Alongside somatic variations, epigenetic variation has been proposed as a pivotal player in regulating phenotypic variability acquired during domestication. The emergence of these epialleles might have significantly influenced grapevine domestication over time. This study aims to investigate the impact of domestication on methylation patterns in cultivated grapevines. Reduced-representation bisulfite sequencing was conducted on 18 cultivated and wild accessions. Results revealed that cultivated grapevines exhibited higher methylation levels than their wild counterparts. Differential Methylation Analysis between wild and cultivated grapevines identified a total of 9955 differentially methylated cytosines, of which 78% were hypermethylated in cultivated grapevines. Functional analysis shows that core methylated genes (consistently methylated in both wild and cultivated accessions) are associated with stress response and terpenoid/isoprenoid metabolic processes. Meanwhile, genes with differential methylation are linked to protein targeting to the peroxisome, ethylene regulation, histone modifications, and defense response. Collectively, our results highlight the significant roles that epialleles may have played throughout the domestication history of grapevines.

DNA methylation in clonal duckweed (Lemna minor L.) lineages reflects current and historical environmental exposures.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Human Induced Pluripotent Stem Cells: From Cell Origin, Genomic Stability, and Epigenetic Memory to Translational Medicine.

The potential of human induced pluripotent stem cells (iPSCs) to self-renew indefinitely and to differentiate virtually into any cell type in unlimited quantities makes them attractive for in vitro disease modeling, drug screening, personalized medicine, and regenerative therapies. As the genome of iPSCs thoroughly reproduces that of the somatic cells from which they are derived, they may possess genetic abnormalities, which would seriously compromise their utility and safety. Genetic aberrations could be present in donor somatic cells and then transferred during iPSC generation, or they could occur as de novo mutations during reprogramming or prolonged cell culture. Therefore, to warrant the safety of human iPSCs for clinical applications, analysis of genetic integrity, particularly during iPSC generation and differentiation, should be carried out on a regular basis. On the other hand, reprogramming of somatic cells to iPSCs requires profound modifications in the epigenetic landscape. Changes in chromatin structure by DNA methylations and histone tail modifications aim to reset the gene expression pattern of somatic cells to facilitate and establish self-renewal and pluripotency. However, residual epigenetic memory influences the iPSC phenotype, which may affect their application in disease therapeutics. The present review discusses the somatic cell origin, genetic stability, and epigenetic memory of iPSCs and their impact on basic and translational research.