Specific targeting of cytosine methylation to DNA sequences in vivo

Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions.     

植物DNA甲基化调控因子研究进展

DNA甲基化是重要的植物基因组表观遗传修饰。植物中DNA甲基化的建立与维持是由多个调控因子协同作用的结果。不同的甲基转移酶类能直接作用于不同位点胞嘧啶甲基化, 其中MET1主要负责保持原初CG位点的甲基化, CMT3主要负责保持CNG位点的甲基化, 并由DRM与CMT3的协同从头甲基化作用来补偿其他相关序列的甲基化。这些甲基转移酶与染色质重塑解旋酶和组蛋白修饰因子协同改变染色质的结构, 行使表观遗传的功能。DNA转葡糖基酶有去甲基化活性从而减轻基因沉默。文章综述了以上植物DNA甲基化调控因子的生物学功能及其之间的相互作用和近年来的研究进展, 以更好的理解DNA甲基化的建立、保持和去除的机制。     

Epigenetic features drastically impact CRISPR–Cas9 efficacy in plants

CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

Epigenetic features substantially influence genome editing efficiency and DNA repair outcomes.     

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.     

Structural and functional features of the 5-methylcytosine distribution in the eukaryotic genome

DNA methylation is an integral part of the mechanism of a remodeling and modification of the chromatin structure. The global complex net of chromatin modification and remodeling reactions is still to be determined, and studies of the mechanisms controlling the epigenetic processes of histone modification and DNA methylation are in their infancy. Cytosine methylation occurs predominantly in CpG sequences of the eukaryotic genome, and it also takes place at symmetric CpHpG and nonsymmetric CpHpH sites (where H is A, T, or C). The modification efficiency of the three types of DNA methylation sites depends on their genomic localization. Different regions of the eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-island shores, differentially methylated regions of imprinted genes, and regions of nonalternative site-specific modification. The three canonical sites (CpG, CpHpG, and CpHpH) differ in DNA methylation efficiency depending on their nucleotide context. An epigenetic code of DNA methylation can be assumed with context differences playing a specific functional role. The review summarizes the main up-to-date data on the structural and functional features of site-specific cytosine methylation in eukaryotic genomes. Pathogenesis-related alterations in the methylation pattern of the eukaryotic genome are considered.     

A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.     

Plant DNA methyltransferases

DNA methylation is an important modification of DNA that plays a role in genome management and in regulating gene expression during development. Methylation is carried out by DNA methyltransferases which catalyse the transfer of a methyl group to bases within the DNA helix. Plants have at least three classes of cytosine methyltransferase which differ in protein structure and function. The METI family, homologues of the mouse Dnmt1 methyltransferase, most likely function as maintenance methyltransferases, but may also play a role in de novo methylation. The chromomethylases, which are unique to plants, may preferentially methylate DNA in heterochromatin; the remaining class, with similarity to Dnmt3 methyltransferases of mammals, are putative de novo methyltransferases. The various classes of methyltransferase may show differential activity on cytosines in different sequence contexts. Chromomethylases may preferentially methylate cytosines in CpNpG sequences while the Arabidopsis METI methyltransferase shows a preference for cytosines in CpG sequences. Additional proteins, for example DDM1, a member of the SNF2/SWI2 family of chromatin remodelling proteins, are also required for methylation of plant DNA.     

The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.     

Nuclear architecture in the light of gene expression and cell differentiation studies

It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their 'epigenomes'. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.     

RNA介导的植物DNA甲基化研究进展

RNA介导的DNA甲基化作用(RNA-directed DNA Methylation,RdDM)是首次在植物中发现的基因组表观修饰现象,RdDM通过RNA-DNA序列相互作用直接导致DNA甲基化。植物中的RdDM和siRNA介导的mRNA降解现象,都是通过RNA使序列特异性基因发生沉默,它们对于植物的染色体重排、抵御病毒感染、基因表达调控和发育的许多过程起到了非常重要的作用。在植物中有很多的文献报道RdDM现象,但是对于其具体调控机理还不是很清楚。这里对RNA介导的植物DNA甲基化的基本特征进行了简要概述,主要对RdDM机理的研究进展进行了综述,其中包括RdDM过程中的DNA甲基转移酶的种类及其作用机理,DNA甲基化与染色质修饰之间的关系,以及与RdDM相关的重要蛋白质的研究等。在植物中,转录和转录后水平都可能发生RdDM,诱发基因沉默,前者常涉及靶基因启动子的甲基化,后者则牵涉到编码区的甲基化。RdDM的发生依赖于RNAi途径中相似的siRNA和酶,如DCL3、RdR2、SDE4和AGO4。植物中至少含有三类DNA甲基转移酶DRM1/2、MET1和CMT3,其作用部位是与RNA同源的DNA区域中的所有胞嘧啶,而组蛋白H3第九位赖氨酸的甲基化影响着胞嘧啶的甲基化。     

The many faces of histone lysine methylation

Diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity. Within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone H3 amino terminus. All known histone methyltransferases of this type contain the evolutionarily conserved SET domain and appear to be able to stimulate either gene repression or gene activation. Methylation of H3 Lys9 and Lys4 has been visualised in native chromatin, indicating opposite roles in structuring repressive or accessible chromatin domains. For example, at the mating-type loci in Schizosaccharomyces pombe, at pericentric heterochromatin and at the inactive X chromosome in mammals, striking differences between these distinct marks have been observed. H3 Lys9 methylation is also important to direct additional epigenetic signals such as DNA methylation--for example, in Neurospora crassa and in Arabidopsis thaliana. Together, the available data strongly establish histone lysine methylation as a central modification for the epigenetic organisation of eukaryotic genomes.