Project description:The histone methyltransferase DOT1L catalyzes methylation of H3K79 and it is highly conserved in mammals. DOT1L plays a functional role in several biological processes including cell cycle regulation, DNA repair, RNA splicing and gene expression, suggesting a complex role in chromatin organization and regulation. Such a remarkable range of functions performed by DOT1L can be the result, at least partially, of its interaction with a plethora of proteins and presence in different complexes. Here, we characterized the cooperation of DOT1L with the nucleolar protein NPM1 and the impact of both proteins on peri-nucleolar heterochromatin activity. We show that i) DOT1L interacts preferentially with monomeric NPM1 in the nucleus; ii) DOT1L acts in concert with NPM1 to maintain each other’s protein homeostasis; iii) NPM1 depletion results in H3K79me2 upregulation at chromatin remodeling genes and transcriptional activation of Ezh2; iv) DOT1L and NPM1 preserved DNA satellite expression at peri-nucleolar heterochromatin via epigenetic mechanisms dependent on H3K27me3. Our findings give insights into molecular mechanisms employed by DOT1L and NPM1 to regulate heterochromatin activities around the nucleoli and shed light on one aspect of the complex role of both proteins in chromatin dynamics.
Project description:The histone methyltransferase DOT1L catalyzes methylation of H3K79 and it is highly conserved in mammals. DOT1L plays a functional role in several biological processes including cell cycle regulation, DNA repair, RNA splicing and gene expression, suggesting a complex role in chromatin organization and regulation. Such a remarkable range of functions performed by DOT1l can be the result, at least partially, of its interaction with different proteins and presence in different complexes. Here, we characterized the interaction of DOT1L with the nucleolar protein NPM1 and the impact of the DOT1L/NPM1 complex on nucleoli organization and activity. We show that i) DOT1L interacts preferentially with monomeric NPM1 in the nucleus; ii) DOT1L interaction with NPM1 is key to maintain their protein homeostasis; iii) NPM1 depletion results in H3K79me2 upregulation at chromatin remodeling genes; iv) DOT1L/NPM1 complex preserved peri-nucleolar heterochromatin organization via epigenetic mechanisms. Our findings unravel the molecular mechanisms employed by the DOT1L/NPM1 complex to maintain heterochromatin organization around the nucleoli and shed light on one aspect of the complex role of DOT1L in chromatin dynamics.
Project description:BackgroundNPM1 is a phosphoprotein highly abundant in the nucleolus. However, additional nuclear functions have been attributed to NPM1, probably through interaction with other nuclear factors. DOT1L is one interaction partner of NPM1 that catalyzes methylation of histone H3 at lysine 79 (H3K79). DOT1L, playing functional roles in several biological processes, is known for its capability to organize and regulate chromatin. For example, DOT1L modulates DNA repeats expression within peri-nucleolar heterochromatin. NPM1 also affects peri-nucleolar heterochromatin spatial organization. However, it is unclear as of yet whether NPM1 and DOT1L functionally synergize to preserve nucleoli organization and genome stability, and generally, which molecular mechanisms would be involved.ResultsWe characterized the nuclear function of NPM1 on peri-nucleolar heterochromatin organization. We show that (i) monomeric NPM1 interacts preferentially with DOT1L in the nucleus; (ii) NPM1 acts in concert with DOT1L to maintain each other's protein homeostasis; (iii) NPM1 depletion results in H3K79me2 upregulation and differential enrichment at chromatin binding genes including Ezh2; (iv) NPM1 and DOT1L modulate DNA repeats expression and peri-nucleolar heterochromatin organization via epigenetic mechanisms dependent on H3K27me3.ConclusionsOur findings give insights into molecular mechanisms employed by NPM1 and DOT1L to regulate heterochromatin activity and structural organization around the nucleoli and shed light on one aspect of the complex role of both proteins in chromatin dynamics.
Project description:Nucleophosmin 1 regulates H3K79me2 levels and peri-nucleolar heterochromatin organization through interaction with the methyltransferase DOT1L
Project description:Nucleophosmin 1 regulates H3K79me2 levels and peri-nucleolar heterochromatin organization through interaction with the methyltransferase DOT1L [ChIP-seq]
Project description:Nucleophosmin 1 regulates H3K79me2 levels and peri-nucleolar heterochromatin organization through interaction with the methyltransferase DOT1L [RNA-seq]
Project description:Epigenetic modification, including histone modification, precisely controls target gene expression. The posttranscriptional regulation of the innate signaling-triggered production of inflammatory cytokines and type I interferons has been fully elucidated, whereas the roles of histone modification alteration and epigenetic modifiers in regulating inflammatory responses need to be further explored. Di/tri-methylation modifications of histone 3 lysine 79 (H3K79me2/3) have been shown to be associated with gene transcriptional activation. Disruptor of telomeric silencing-1-like (Dot1l) is the only known exclusive H3K79 methyltransferase and regulates the proliferation and differentiation of tumor cells. However, the roles of Dot1l and Dot1l-mediated H3K79 methylation in innate immunity and inflammatory responses remain unclear. Here, we found that H3K79me2/3 modification levels at the Il6 and Ifnb1 promoters, as well as H3K79me2 modification at the Tnf? promoter, were increased in macrophages activated by Toll-like receptor (TLR) ligands or virus infection. The innate signals upregulated Dot1l expression in macrophages and THP1 cells. Dot1l silencing or a Dot1l inhibitor preferentially suppressed the production of IL-6 and interferon (IFN)-? but not of TNF-? in macrophages and THP1 cells triggered by TLR ligands or virus infection. Dot1l was recruited to the proximal promoter of the Il6 and Ifnb1 but not Tnf? gene and then mediated H3K79me2/3 modification at the Il6 and Ifnb1 promoters, consequently facilitating the transcription and expression of Il6 and Ifnb1. Thus, Dot1l-mediated selective H3K79me2/3 modifications at the Il6 and Ifnb1 promoters are required for the full activation of innate immune responses. This finding adds new insights into the epigenetic regulation of inflammatory responses and pathogenesis of autoimmune diseases.
Project description:Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.
Project description:Epigenetic changes on DNA and chromatin are implicated in cell differentiation and organogenesis. For the heart, distinct histone methylation profiles were recently linked to stage-specific gene expression programs during cardiac differentiation in vitro. However, the enzymes catalyzing these modifications and the genes regulated by them remain poorly defined. We therefore decided to identify the epigenetic enzymes that are potentially involved in cardiomyogenesis by analyzing the expression profile of the 85 genes encoding the epigenetic-related proteins in mouse cardiomyocytes (CMs), and then study how they affect gene expression during differentiation and maturation of this cell type. We show here with gene expression screening of epigenetic enzymes that the highly expressed H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) drives a transitional pattern of di-methylation on H3 lysine 79 (H3K79) in CMs at different stages of differentiation in vitro and in vivo. Through a genome-wide chromatin-immunoprecipitation DNA-sequencing approach, we found H3K79me2 enriched at genes expressed during cardiac differentiation. Moreover, knockdown of Dot1L affected the expression of H3K79me2-enriched genes. Our results demonstrate that histone methylation, and in particular DOT1L-mediated H3K79me2 modification, drives cardiomyogenesis through the definition of a specific transcriptional landscape.
Project description:Mercury is a highly toxic element that induces severe alterations and a broad range of adverse effects on health. Its exposure is a global concern because it is widespread in the environment due to its multiple industrial, domestic, agricultural and medical usages. Among its various chemical forms, both humans and animals are mainly exposed to mercury chloride (HgCl2), methylmercury and elemental mercury. HgCl2 is metabolized primarily in the liver. We analysed the effects on the nuclear architecture of an increasing dosage of HgCl2 in mouse hepatocytes cell culture and in mouse liver, focusing specifically on the organization, on some epigenetic features of the heterochromatin domains and on the nucleolar morphology and activity. Through the combination of molecular and imaging approaches both at optical and electron microscopy, we show that mercury chloride induces modifications of the heterochromatin domains and a decrease of some histones post-translational modifications associated to heterochromatin. This is accompanied by an increase in nucleolar activity which is reflected by bigger nucleoli. We hypothesized that heterochromatin decondensation and nucleolar activation following mercury chloride exposure could be functional to express proteins necessary to counteract the harmful stimulus and reach a new equilibrium.