Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis

SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants mono- and dimethyl H3K9, mono- and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

The SRA methyl-cytosine-binding domain links DNA and histone methylation

Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.     

Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3

Both DNA methylation and post-translational histone modifications contribute to gene silencing, but the mechanistic relationship between these epigenetic marks is unclear. Mutations in two Arabidopsis genes, the KRYPTONITE (KYP) histone H3 lysine 9 (H3K9) methyltransferase and the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase, cause a reduction of CNG DNA methylation, suggesting that H3K9 methylation controls CNG DNA methylation. Here we show that the chromodomain of CMT3 can directly interact with the N-terminal tail of histone H3, but only when it is simultaneously methylated at both the H3K9 and H3K27 positions. Furthermore, using chromatin immunoprecipitation analysis and immunohistolocalization experiments, we found that H3K27 methylation colocalizes with H3K9 methylation at CMT3-controlled loci. The H3K27 methylation present at heterochromatin was not affected by mutations in KYP or in several Arabidopsis PcG related genes including the Enhancer of Zeste homologs, suggesting that a novel pathway controls heterochromatic H3K27 methylation. Our results suggest a model in which H3K9 methylation by KYP, and H3K27 methylation by an unknown enzyme provide a combinatorial histone code for the recruitment of CMT3 to silent loci.     

An Arabidopsis SET domain protein required for maintenance but not establishment of DNA methylation

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation-correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis: This screen yielded seven loss-of-function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2, especially in non-CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.     

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.     

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (～E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.     

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

Investigations aimed at identifying regulators of nuclear architecture in Drosophila demonstrated that cells lacking H3K9 methylation and RNA interference (RNAi) pathway components displayed disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared with wild type. We also observed a substantial increase in extrachromosomal circular (ecc) repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 and ecc DNA formation is not induced by removal of cohesin. We conclude that the structural integrity and organization of repeated DNAs and nucleoli are regulated by the H3K9 methylation and RNAi pathways, and other regulators of heterochromatin-mediated silencing. In addition, repeated DNA stability involves suppression of non-homologous end joining (NHEJ) or other recombination pathways. These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.