RNAi-independent de novo DNA methylation revealed in Arabidopsis mutants of chromatin remodeling gene DDM1

Methylation of histone H3 lysine 9 (H3K9me) and small RNAs are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non‐CpG sites. Transposon‐specific non‐CpG methylation in plants is controlled by small RNAs and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly demonstrated in plants. We have previously shown that a mutation in the chromatin remodeling gene DDM1 (DECREASE IN DNA METHYLATION 1) induces a global decrease but a local increase of cytosine methylation and accumulation of small RNA at a locus called BONSAI. Here we show that de novo BONSAI methylation does not depend on RNAi but does depend on H3K9me. In mutants of H3K9 methyltransferase gene KRYPTONITE or the H3K9me‐dependent DNA methyltransferase gene CHROMOMETHYALSE3, the ddm1‐induced de novo cytosine methylation was abolished for all three contexts (CpG, CpHpG and CpHpH). Furthermore, RNAi mutants showed strong developmental defects when combined with the ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications that may be conserved among diverse eukaryotes.     

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.     

Pairing of <Emphasis Type="Italic">lacO</Emphasis> tandem repeats in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> nuclei requires the presence of hypermethylated,large arrays at two chromosomal positions,but does not depend on H3-lysine-9-dimethylation

Fluorescent chromatin tagging by the lacO operator/lac repressor system in Arabidopsis thaliana is useful to trace distinct chromatin domains in living cells. Nevertheless, the tandem repeats of the tagging system may alter the spatial organisation of chromatin within nuclei by increasing homologous pairing as well as association with heterochromatin. Efficient homologous pairing occurs if lacO repeat arrays of ∼10 kb are present at two loci, either on the same chromosome or on different chromosomes. DNA hypomethylation of lacO repeats results in reduced homologous pairing. Because, in plants, DNA methylation can serve as a signal for H3-lysine9-dimethylation (H3K9me2), and subsequently for non-CG-context DNA methylation, SET-domain histone methyltransferase and chromodomain dna methyltransferase 3 (cmt3) mutations were introgressed. In suvh4 suvh5 suvh6 and cmt3 mutants, H3K9me2 associated with lacO repeats is diminished, but homologous pairing persists. Thus, neither H3K9me2 nor CMT3-mediated non-CG methylation are required at wild-type level for homologous pairing of lacO repeat loci.     

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A–C and JMJD2A–D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.     

Recognition of H3K9me1 by maize RNA-directed DNA methylation factor SHH2

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation pathway, which has extensive cross-talk with histone modifications. Here, we report that the maize RdDM regulator SAWADEE HOMEODOMAIN HOMOLOG 2 (SHH2) is an H3K9me1 reader. Our structural studies reveal that H3K9me1 recognition is achieved by recognition of the methyl group via a classic aromatic cage and hydrogen-bonding and salt-bridge interactions with the free protons of the mono-methyllysine. The di- and tri-methylation states disrupt the polar interactions, decreasing the binding affinity. Our study reveals a mono-methyllysine recognition mechanism which potentially links RdDM to H3K9me1 in maize.