H3 Variant Research Articles

CNSC-36. REGULATION OF CELL FATE IN PAEDIATRIC HIGH-GRADE GLIOMA BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Paediatric high-grade gliomas (pHGG) are nearly uniformly fatal cancers that arise predominantly in children and adolescents and encompass Diffuse Midline Glioma (DMG) and Diffuse Hemispheric Glioma (DHG). Most patients harbour mutations in histone variants H3.1 or H3.3, resulting in global epigenetic dysregulation and a stalled oligodendroglial progenitor (OPC) or GABAergic interneuron progenitor (IP) fate for DMG and DHG, respectively. DLX2, a homeobox transcription factor, represses OPC fate and promotes IP cell fate and migration during early neurogenesis. Our lab has shown that Dlx2 binds to and regulates the promoters of Gad1/Gad2, as well as early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of Dlx2 with these target sequences reduces reporter gene expression in vitro and Dlx1/2 double knockout mice exhibit increased expression of these OPC markers in vivo. Transient overexpression of a Dlx2-GFP construct in murine DMG cells led to significant IP marker increase (Gad1/2), decrease of OPC markers (Olig2, Nkx2.2) and global restoration of H3K27me3. Additionally, decreased proliferation, colony growth, invasion and migration were observed in vitro. To translate these findings in human pHGG, analysis of a bulk RNA-seq database of 62 patient-derived pHGG cell lines revealed an inverse relationship between DLX2 and OLIG2. DLX2 was knocked out of DLX2high/OLIG2low DHG cell lines using CRISPR-Cas9, and overexpressed in DLX2low/OLIG2high DMG cell lines using a DLX2-mCherry overexpression construct. Evaluation of cell fate markers and epigenetic marks will be presented. Changes in migration, invasion, and proliferation in vitro will also be presented, in addition to changes in tumour growth using a neonatal xenograft murine model in vivo. Manipulation of DLX2 in pHGG provides a model for investigating the link between the factors that control cell fate decisions in the developing brain and the aberrant differentiation state in pHGG.

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa.

A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.

Dinochromosome Heterotermini with Telosomal Anchorages

Dinoflagellate birefringent chromosomes (BfCs) contain some of the largest known genomes, yet they lack typical nucleosomal micrococcal-nuclease protection patterns despite containing variant core histones. One BfC end interacts with extranuclear mitotic microtubules at the nuclear envelope (NE), which remains intact throughout the cell cycle. Ultrastructural studies, polarized light and fluorescence microscopy, and micrococcal nuclease-resistant profiles (MNRPs) revealed that NE-associated chromosome ends persisted post-mitosis. Histone H3K9me3 inhibition caused S-G2 delay in synchronous cells, without any effects at G1. Differential labeling and nuclear envelope swelling upon decompaction indicate an extension of the inner compartment into telosomal anchorages (TAs). Additionally, limited effects of low-concentration sirtinol on bulk BfCs, coupled with distinct mobility patterns in MNase-digested and psoralen-crosslinked nuclei observed on 2D gels, suggest that telomeric nucleosomes (TNs) are the primary histone structures. The absence of a nucleosomal ladder with cDNA probes, the presence of histone H2A and telomere-enriched H3.3 variants, along with the immuno-localization of H3 variants mainly at the NE further reinforce telomeric regions as the main nucleosomal domains. Cumulative biochemical and molecular analyses suggest that telomeric repeats constitute the major octameric MNRPs that provision chromosomal anchorage at the NE.

Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading.

Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions betwen MSL3 (male-specific lethal 3) and Set2-dependent histone marks like trimethylated H3 lysine-36 (H3K36me3). However, Set2 could affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Furthermore, it is important to parse male-specific effects from those that are X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 'residue' and Set2 'writer' mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global, male-specific reduction of X-genes in Set2 or H3K36 mutants, we observe heterogeneous effects. Interestingly, we identified groups of differentially expressed genes (DEGs) whose changes were in opposite directions following loss of H3K36 or Set2, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, differential expression analysis of combined H3.2K36R/H3.3K36R mutants showed neither consistent reduction in X-gene expression, nor correlation with MSL3 binding. Motif analysis of the DEGs implicated BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 is essential for spreading the MSL complex to genes along the male X. Rather, we propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

The white gene as a transgenesis marker for the cricket Gryllus bimaculatus.

The cricket Gryllus bimaculatus is an emerging model insect of the order Orthoptera that is used in a wide variety of biological research themes. This hemimetabolous species appears highly complementary to Drosophila and other well-established holometabolous models. To improve transgenesis applications in G. bimaculatus, we have designed a transformation marker gene inspired from the widespread Drosophila mini-white+. Using CRISPR/Cas9, we first generated a loss-of-function mutant allele of the Gb-white gene (Gb-w), which exhibits a white eye coloration at all developmental stages. We then demonstrate that transgenic insertions of a piggyBac vector containing a 3xP3-Gb-w+ cassette rescue eye pigmentation. As an application, we used this vector to generate G. bimaculatus lines expressing a centromeric histone H3 variant (CenH3.1) fused to EGFP and validated EGFP-CenH3.1 detection at cricket centromeres. Finally, we demonstrate that Minos-based germline transformation and site-specific plasmid insertion with the ΦC31 integrase system function in G. bimaculatus.

Structure and evolution of metapolycentromeres.

Metapolycentromeres consist of multiple sequential domains of centromeric chromatin associated with a centromere-specific variant of histone H3 (CENP-A), functioning collectively as a single centromere. To date, they have been revealed in nine flowering plant, five insect and six vertebrate species. In this paper, we focus on their structure and possible mechanisms of emergence and evolution. The metapolycentromeres may vary in the number of centromeric domains and in their genetic content and epigenetic modifications. However, these variations do not seem to affect their function. The emergence of metapolycentromeres has been attributed to multiple Robertsonian translocations and segmental duplications. Conditions of genomic instability, such as interspecific hybridization and malignant neoplasms, are suggested as triggers for the de novo emergence of metapolycentromeres. Addressing the "centromere paradox" - the rapid evolution of centromeric DNA and proteins despite their conserved cellular function - we explore the centromere drive hypothesis as a plausible explanation for the dynamic evolution of centromeres in general, and in particular the emergence of metapolycentromeres and holocentromeres. Apparently, metapolycentromeres are more common across different species than it was believed until recently. Indeed, a systematic review of the available cytogenetic publications allowed us to identify 27 candidate species with metapolycentromeres. Тhe list of the already established and newly revealed candidate species thus spans 27 species of flowering plants and eight species of gymnosperm plants, five species of insects, and seven species of vertebrates. This indicates an erratic phylogenetic distribution of the species with metapolycentromeres and may suggest an independent emergence of the metapolycentromeres in the course of evolution. However, the current catalog of species with identified and likely metapolycentromeres remains too short to draw reliable conclusions about their evolution, particularly in the absence of knowledge about related species without metapolycentromeres for comparative analysis. More studies are necessary to shed light on the mechanisms of metapolycentromere formation and evolution.

A Site-Specific Click Chemistry Approach to Di-ubiquitylate H1 Variants Reveals Position-dependent Stimulation of the DNA Repair Protein RNF168.

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click-chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection.

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a ΔCTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection.

Spatial patterns of influenza A virus spread across compartments in commercial swine farms in the United States.

Multiple genetic variants of H1 and H3 influenza A viruses (IAVs) circulate concurrently in US swine farms. Understanding the spatial transmission patterns of IAVs among these farms is crucial for developing effective control strategies and mitigating the emergence of novel IAVs. In this study, we analysed 1909 IAV genomic sequences from 785 US swine farms, representing 33 farming systems across 12 states, primarily in the Midwest from 2004 to 2023. Bayesian phylogeographic analyses were performed to identify the dispersal patterns of both H1 and H3 virus genetic lineages and to elucidate their spatial migration patterns within and between different systems. Our results showed that both intra-system and inter-system migrations occurred between the swine farms, with intra-system migrations being more frequent. However, migration rates for H1 and H3 IAVs were similar between intra-system and inter-system migration events. Spatial migration patterns aligned with expected pig movement across different compartments of swine farming systems. Sow-Farms were identified as key sources of viruses, with bi-directional migration observed between these farms and other parts of the system, including Wean-to-Finish and Gilt-Development-Units. High intra-system migration was detected across farms in the same region, while spread to geographically distant intra- and inter-system farms was less frequent. These findings suggest that prioritizing resources towards systems frequently confronting influenza problems and targeting pivotal source farms, such as sow farms, could be an effective strategy for controlling influenza in US commercial swine operations.

Linker histones H1 in human disease

Linker histones (H1) are basic proteins that are part of the nucleosome structure in the cell nucleus and are involved in the packaging of genetic material and the regulation of gene expression. As research progressed, it was discovered that linker histones constitute the largest group of histones in terms of variants found in humans. Even though the H1 variants differ slightly in the primary structure, they can perform different functions, undergo multiple post-translational modifications and differ in cellular localization. In addition to the nucleus, histones H1 can occur in the cytoplasm, on the cell surface and in the intercellular space. In these places, they play a supporting role for the immune system and act as signaling molecules. Changes in the levels of histones and their post-translational modifications have been associated with many human diseases and it is postulated that some of them may serve as biomarkers or therapeutic targets.

M6A-modified cenRNA stabilizes CENPA to ensure centromere integrity in cancer cells

m6A modification is best known for its critical role in controlling multiple post-transcriptional processes of the mRNAs. Here, we discovered elevated levels of m6A modification on centromeric RNA (cenRNA) in cancerous cells compared with non-cancerous cells. We then identified CENPA, an H3 variant, as an m6A reader of cenRNA. CENPA is localized at centromeres and is essential in preserving centromere integrity and function during mitosis. The m6A-modified cenRNA stabilizes centromeric localization of CENPA in cancer cells during the S phase of the cell cycle. Mutations of CENPA at the Leu61 and the Arg63 or removal of cenRNA m6A modification lead to loss of centromere-bound CENPA during S phase. This in turn results in compromised centromere integrity and abnormal chromosome separation and hinders cancer cell proliferation and tumor growth. Our findings unveil an m6A reading mechanism by CENPA that epigenetically governs centromere integrity in cancer cells, providing potential targets for cancer therapy.

Argonaute protein CSR-1 restricts localization of holocentromere protein HCP-3, the C. elegans CENP-A homolog.

Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos.

Age-related histone H3.3 accumulation associates with a repressive chromatin in mouse tibialis anterior muscle

The present study aimed to investigate age-related changes in histone variant H3.3 and its role in the aging process of mouse tibialis anterior muscle. H3.3 level significantly increased with age and correlated with H3K27me3 level. Acute exercise successfully upregulated the target gene expression in 8-wk-old mice, whereas no upregulation was noted in 53-wk-old mice. H3K27me3 level was increased at these loci in response to acute exercise in 8-wk-old mice. However, in 53-wk-old mice, H3.3 and H3K27me3 levels were increased at rest and were not affected by acute exercise. Furthermore, forced H3.3 expression in the skeletal muscle of 8-wk-old mice led to a gradual improvement in motor function. The results suggest that age-related H3.3 accumulation induces the formation of repressive chromatin in the mouse tibialis anterior muscle. However, H3.3 accumulation also appears to play a positive role in enhancing skeletal muscle function.

Centromere inactivation during aging can be rescued in human cells.

Aging involves a range of genetic, epigenetic, and physiological alterations. A key characteristic of aged cells is the loss of global heterochromatin, accompanied by a reduction in canonical histone levels. In this study, we track the fate of centromeres during aging in human cells. Our findings reveal that the centromeric histone H3 variant CENP-A is downregulated in aged cells, in a p53-dependent manner. We observe repression of centromeric noncoding transcription through an epigenetic mechanism via recruitment of a lysine-specific demethylase 1 (LSD1/KDM1A) to centromeres. This suppression results in defective de novo CENP-A loading at aging centromeres. By dual inhibition of p53 and LSD1/KDM1A in aged cells, we mitigate the reduction in centromeric proteins and centromeric transcripts, leading to mitotic rejuvenation of these cells. These results offer insights into a novel mechanism for centromeric inactivation during aging and provide potential strategies to reactivate centromeres.

Roles of Histone H2B, H3 and H4 Variants in Cancer Development and Prognosis.

Histone variants are the paralogs of core histones (H2A, H2B, H3 and H4). They are stably expressed throughout the cell cycle in a replication-independent fashion and are capable of replacing canonical counterparts under different fundamental biological processes. Variants have been shown to take part in multiple processes, including DNA damage repair, transcriptional regulation and X chromosome inactivation, with some of them even specializing in lineage-specific roles like spermatogenesis. Several reports have recently identified some unprecedented variants from different histone families and exploited their prognostic value in distinct types of cancer. Among the four classes of canonical histones, the H2A family has the greatest number of variants known to date, followed by H2B, H3 and H4. In our prior review, we focused on summarizing all 19 mammalian histone H2A variants. Here in this review, we aim to complete the full summary of the roles of mammalian histone variants from the remaining histone H2B, H3, and H4 families, along with an overview of their roles in cancer biology and their prognostic value in a clinical context.

H3.3K122A results in a neomorphic phenotype in mouse embryonic stem cells.

The histone variant H3.3 acts in coordination with histone posttranslational modifications and other chromatin features to facilitate appropriate transcription. Canonical histone H3 and histone variant H3.3 are post-translationally modified with the genomic distribution of these marks denoting different features and with more recent evidence suggesting that these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the residue H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent, albeit with reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue of H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells.

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription.

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

β-TrCP-Mediated Proteolysis of Mis18β Prevents Mislocalization of CENP-A and Chromosomal Instability.

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.

Clinical immunity to malaria involves epigenetic reprogramming of innate immune cells.

The regulation of inflammation is a critical aspect of disease tolerance and naturally acquired clinical immunity to malaria. Here, we demonstrate using RNA sequencing and epigenetic landscape profiling by cytometry by time-of-flight, that the regulation of inflammatory pathways during asymptomatic parasitemia occurs downstream of pathogen sensing-at the epigenetic level. The abundance of certain epigenetic markers (methylation of H3K27 and dimethylation of arginine residues) and decreased prevalence of histone variant H3.3 correlated with suppressed cytokine responses among monocytes of Ugandan children. Such an epigenetic signature was observed across diverse immune cell populations and not only characterized active asymptomatic parasitemia but also correlated with future long-term disease tolerance and clinical immunity when observed in uninfected children. Pseudotime analyses revealed a potential trajectory of epigenetic change that correlated with a child's age and recent parasite exposure and paralleled the acquisition of clinical immunity. Thus, our data support a model whereby exposure to Plasmodium falciparum induces epigenetic changes that regulate excessive inflammation and contribute to naturally acquire clinical immunity to malaria.