Binding specificity of ASHH2 CW-domain towards H3K4me1 ligand is coupled to its structural stability through its α1-helix

The CW domain binds to histone-tail modifications found in different protein families involved in epigenetic regulation and chromatin remodelling. CW domains recognize the methylation state of the fourth lysine on histone 3, and as such could be viewed as a reader of epigentic information. The specificity towards different methylation states such as me1, me2 or me3 depend on the particular subtype. For example, the CW domain of ASHH2-methyltransferase binds preferentially to H3K4me1, MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings are not understood well, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity towards H3K4me1, and the stabilization of the domain. Key elements of the binding site are the two tryptophans and the a1-helix form and maintain the binding pocket were perturbed by mutagenesis and investigated. Results show that a1-helix maintains the overall stability of the fold via the I915 and L919 residues, and that correct binding consolidates the coils designated n1, n3, as well a the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability upon binding. Moreover, loop-mutations not directly involved in the binding site nonetheless affect the equilibrium positions of key residues.

The genomic loci of specific human tRNA genes exhibit ageing-related DNA hypermethylation

The epigenome has been shown to deteriorate with age, potentially impacting on ageing-related disease. tRNA, while arising from only ˜46 kb (<0.002% genome), is the second most abundant cellular transcript. tRNAs also control metabolic processes known to affect ageing, through core translational and additional regulatory roles. Here, we interrogate the DNA methylation state of the genomic loci of human tRNA. We identify a genomic enrichment for age-related DNA hypermethylation at tRNA loci. Analysis in 4,350 MeDIP-seq peripheral-blood DNA methylomes (16–82 years), identifies 44 and 21 hypermethylating specific tRNAs at study-and genome-wide significance, respectively, contrasting with none hypomethylating. Validation and replication (450k array and independent targeted Bisuphite-sequencing) supported the hypermethylation of this functional unit. Tissue-specificity is a significant driver, although the strongest consistent signals, also independent of major cell-type change, occur in tRNA-iMet-CAT-1-4 and tRNA-Ser-AGA-2-6. This study presents a comprehensive evaluation of the genomic DNA methylation state of human tRNA genes and reveals a discreet hypermethylation with advancing age.

Lifetime genealogical divergence within plants leads to epigenetic mosaicism in the long lived shrub Lavandula latifolia (Lamiaceae)

Epigenetic mosaicism is a possible source of within-plant phenotypic heterogeneity, yet its frequency and developmental origin remain unexplored. This study examines whether the extant epigenetic heterogeneity within long-lived Lavandula latifolia (Lamiaceae) shrubs reflects recent epigenetic modifications experienced independently by different plant parts or, alternatively, it is the cumulative outcome of a steady lifetime process. Leaf samples from different architectural modules were collected from three L. latifolia plants and characterized epigenetically by global DNA cytosine methylation and methylation state of methylation-sensitive amplified fragment length polymorphism markers (MS-AFLP). Epigenetic characteristics of modules were then assembled with information on the branching history of plants. Methods borrowed from phylogenetic research were used to assess genealogical signal of extant epigenetic variation and reconstruct within-plant genealogical trajectory of epigenetic traits. Plants were epigenetically heterogeneous, as shown by differences among modules in global DNA methylation and variation in the methylation states of 6-8% of MS-AFLP markers. All epigenetic features exhibited significant genealogical signal within plants. Events of epigenetic divergence occurred throughout the lifespan of individuals and were subsequently propagated by branch divisions. Internal epigenetic diversification of L. latifolia individuals took place steadily during their development, a process which eventually led to persistent epigenetic mosaicism.

A DNA methylation state transition model reveals the programmed epigenetic heterogeneity in human pre-implantation embryos

Background During mammalian early embryogenesis, expression and epigenetic heterogeneity emerge before the first cell fate determination, but the programs causing such determinate heterogeneity are largely unexplored. Results Here, we present MethylTransition, a novel DNA methylation state transition model, for characterizing methylation changes during one or a few cell cycles at single-cell resolution. MethylTransition involves the creation of a transition matrix comprising three parameters that represent the probabilities of DNA methylation-modifying activities in order to link the methylation states before and after a cell cycle. We apply MethylTransition to single-cell DNA methylome data from human pre-implantation embryogenesis and elucidate that the DNA methylation heterogeneity that emerges at promoters during this process is largely an intrinsic output of a program with unique probabilities of DNA methylation-modifying activities. Moreover, we experimentally validate the effect of the initial DNA methylation on expression heterogeneity in pre-implantation mouse embryos. Conclusions Our study reveals the programmed DNA methylation heterogeneity during human pre-implantation embryogenesis through a novel mathematical model and provides valuable clues for identifying the driving factors of the first cell fate determination during this process.

Detection of haplotype-dependent allele-specific DNA methylation in WGBS data

In heterozygous genomes, allele-specific measurements can reveal biologically significant differences in DNA methylation between homologous alleles associated with local changes in genetic sequence. Current approaches for detecting such events from whole-genome bisulfite sequencing (WGBS) data perform statistically independent marginal analysis at individual cytosine-phosphate-guanine (CpG) sites, thus ignoring correlations in the methylation state, or carry-out a joint statistical analysis of methylation patterns at four CpG sites producing unreliable statistical evidence. Here, we employ the one-dimensional Ising model of statistical physics and develop a method for detecting allele-specific methylation (ASM) events within segments of DNA containing clusters of linked single-nucleotide polymorphisms (SNPs), called haplotypes. Comparisons with existing approaches using simulated and real WGBS data show that our method provides an improved fit to data, especially when considering large haplotypes. Importantly, the method employs robust hypothesis testing for detecting statistically significant imbalances in mean methylation level and methylation entropy, as well as for identifying haplotypes for which the genetic variant carries significant information about the methylation state. As such, our ASM analysis approach can potentially lead to biological discoveries with important implications for the genetics of complex human diseases.

Selective Binding of a Lower Lysine Methylation State: An N,N-Dimethyllysine Selective Host Molecule and Its Use in Methyl Proteomics

<p>Post-translational modifications (PTMs) are critical controllers of protein functions. One set of important PTMs are <i>N</i>-methylated side chains of lysine and arginine, which exist in several functionally distinct forms. Multiple groups have demonstrated the selective binding of the most hydrophobic family member, trimethyllysine (Kme3), using various macrocyclic hosts, but the selective binding of lower methylation states remains challenging. Herein we report that a new calixarene modification – the installation of a sulfonate ester at the lower rim of <i>p</i>-sulfonatocalix[4]arene —efficiently generates a <i>N,N</i>-dimethyllysine (Kme2)-selective host. We characterize its binding behaviors in solution, and demonstrate its effectiveness in a pan-methyllysine enrichment step that enables the observation of hundreds of otherwise unobservable methylation marks in global proteomics experiments.</p><p>The submission includes a manuscript preprint, supporting information, and a tabulation of proteomics data.</p>

Selective Binding of a Lower Lysine Methylation State: An N,N-Dimethyllysine Selective Host Molecule and Its Use in Methyl Proteomics

<p>Post-translational modifications (PTMs) are critical controllers of protein functions. One set of important PTMs are <i>N</i>-methylated side chains of lysine and arginine, which exist in several functionally distinct forms. Multiple groups have demonstrated the selective binding of the most hydrophobic family member, trimethyllysine (Kme3), using various macrocyclic hosts, but the selective binding of lower methylation states remains challenging. Herein we report that a new calixarene modification – the installation of a sulfonate ester at the lower rim of <i>p</i>-sulfonatocalix[4]arene —efficiently generates a <i>N,N</i>-dimethyllysine (Kme2)-selective host. We characterize its binding behaviors in solution, and demonstrate its effectiveness in a pan-methyllysine enrichment step that enables the observation of hundreds of otherwise unobservable methylation marks in global proteomics experiments.</p><p>The submission includes a manuscript preprint, supporting information, and a tabulation of proteomics data.</p>

The Genomic Loci of Specific Human tRNA Genes Exhibit Ageing-Related DNA Hypermethylation

The epigenome deteriorates with age, potentially impacting on ageing-related disease.Here, we interrogate the DNA methylation state of the genomic loci of human tRNA. Whilst arising from only ~46kb (<0.002% genome), this information transfer machinery is the second most abundant cellular transcript. tRNAs also control metabolic processes known to affect ageing, through core translational and additional regulatory roles.We identified a genomic enrichment for age-related DNA hypermethylation at tRNA loci. Analysis in 4,350 MeDIP-seq peripheral-blood DNA methylomes (16-82 years), classified 44 and 21 hypermethylating specific tRNAs at study- and genome-wide significance, respectively, contrasting with 0 hypomethylating. Validation and replication (450k array & independent targeted Bisuphite-sequencing) supported thehypermethylation of this functional unit. The strongest consistent signals, also independent of major cell-type change, occur in tRNA-iMet-CAT-1-4 and tRNA-Ser-AGA-2-6.This study is this first comprehensive evaluation of the genomic DNA methylation state of human tRNA genes and reveals a discreet hypermethylation with advancing age.

PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17–92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.