Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1

Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.     

The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix

Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.     

Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain. Here we show, with the use of crystallography, that the carboxy-terminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.     

F-MSAP： A practical system to detect methylation in chicken genome

By replacing radiation with fluorescent system in the technique of methylation sensitive amplified polymorphism （MSAP） and optimizing reaction conditions, a modified technique to detect DNA methylation called F-MSAP （fluorescent labeled methylation sensitive amplified polymorphism） was developed. In the present study, cytosine methylation patterns of genomic DNA were investigated in two inbred chickens and their F1 hybrids. Three types of methylation patterns were observed in each individual, namely fully methylated, hemi-methylated or not methylated types. The average incidence of methylation was approximately 40%. The percentage that the F1 hybrid individual inherits the methylation for any given sites from either/both parent amounted to 95%, while the percentage of altered methylation patterns in F1 individual was only 5%, including 14 increased and 12 decreased methylation types, demonstrating that F-MSAP was highly efficient for large-scale detection of cytosine methylation in chicken genome. Our technique can be further extended to other animals or plants with complex genome and rich in methylation polymorphism.     

A unique regulatory phase of DNA methylation in the early mammalian embryo

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)--including many CpG island promoters--that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.     

Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L

Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.     

The methylated component of the Neurospora crassa genome

Cytosine methylation is common, but not ubiquitous, in eukaryotes. Mammals and the fungus Neurospora crassa have about 2-3% of cytosines methylated. In mammals, methylation is almost exclusively in the under-represented CpG dinucleotides, and most CpGs are methylated whereas in Neurospora, methylation is not preferentially in CpG dinucleotides and the bulk of the genome is unmethylated. DNA methylation is essential in mammals but is dispensable in Neurospora, making this simple eukaryote a favoured organism in which to study methylation. Recent studies indicate that DNA methylation in Neurospora depends on one DNA methyltransferase, DIM-2 (ref. 6), directed by a histone H3 methyltransferase, DIM-5 (ref. 7), but little is known about its cellular and evolutionary functions. As only four methylated sequences have been reported previously in N. crassa, we used methyl-binding-domain agarose chromatography to isolate the methylated component of the genome. DNA sequence analysis shows that the methylated component of the genome consists almost exclusively of relics of transposons that were subject to repeat-induced point mutation--a genome defence system that mutates duplicated sequences.     

Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation

Differential modifications of the genome during gametogenesis result in a functional difference between the paternal and maternal genomes at the moment of fertilization. A possible cause of this imprinting is the methylation of DNA. The insertion of foreign DNA into transgenic mice allows the tagging of regions that are differentially methylated during gametogenesis. We describe here a transgenic mouse strain in which the expression of the hepatitis B surface antigen gene is irreversibly repressed following its passage through the female germ line. This inhibition is accompanied by the methylation of all the HpaII and HhaI sites within the foreign gene, which we have shown to be integrated into a site on chromosome 13. The irreversibility reported here contrasts with what is found with other transgenic mice sequences which are reversibly methylated after passage through the male or female germ line, though in both cases methylation appears to be important in the imprinting process.     

Demethylation of CpG islands in embryonic cells

DNA in differentiated somatic cells has a fixed pattern of methylation, which is faithfully copied after replication. By contrast, the methylation patterns of many tissue-specific and some housekeeping genes are altered during normal development. This modification of DNA methylation in the embryo has also been observed in transgenic mice and in transfection experiments. Here we report the fate in mice of an in vitro-methylated adenine phosphoribosyltransferase transgene. The entire 5' CpG island region became demethylated, whereas the 3' end of the gene remained modified and was even methylated de novo at additional sites. Transfection experiments in vitro show that the demethylation is rapid, is specific for embryonic cell-types and affects a variety of different CpG island sequences. This suggests that gene sequences can be recognized in the early embryo and imprinted with the correct methylation pattern through a combination of demethylation and de novo methylation.     

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.     

Predicted methylation landscape of all CpG islands on the human genome

CpG island methylation plays important role in various biological processes. To investigate methylation landscape of all CpG islands on the human genome, we develop a model for predicting the CpG island methylation status. This model outperforms other existing methods. We apply the model on the whole human genome and predict the landscape of DNA methylation of all CpG islands. Based on the methylation profile, we find that about 31% of CpG islands are methylation-prone and CpG islands located in promoter regions are seldom methylated. There is no significant difference in the CpG island methylation level between R and G bands among the chromosomes. The occupancy of RNA polymerase II is significantly higher in methylation-resistant promoter CpG islands, indicating that genes with such promoter CpG islands tend to be more active.     

The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites.

In addition to its well-documented effects on gene silencing, cytosine methylation is a prominent cause of mutations. In humans, the mutation rate from 5-methylcytosine (m5C) to thymine (T) is 10-50-fold higher than other transitions and the methylated sequence CpG is consequently under-represented. Over one-third of germline point mutations associated with human genetic disease and many somatic mutations leading to cancer involve loss of CpG. The primary cause of mutability appears to be hydrolytic deamination. Cytosine deamination produces mismatched uracil (U), which can be removed by uracil glycosylase, whereas m5C deamination generates a G x T mispair that cannot be processed by this enzyme. Correction of m5CpG x TpG mismatches may instead be initiated by the thymine DNA glycosylase, TDG. Here we show that MBD4, an unrelated mammalian protein that contains a methyl-CpG binding domain, can also efficiently remove thymine or uracil from a mismatches CpG site in vitro. Furthermore, the methyl-CpG binding domain of MBD4 binds preferentially to m5CpG x TpG mismatches-the primary product of deamination at methyl-CpG. The combined specificities of binding and catalysis indicate that this enzyme may function to minimize mutation at methyl-CpG.     

DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.     

Genome-scale DNA methylation maps of pluripotent and differentiated cells

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.     

基于病例对照组的检验差异甲基化基因功能区域的方法

DNA甲基化是一种重要的表观遗传学修饰,能够参与基因表达调控从而影响生理活动.在病例-对照组研究中,利用甲基化水平定位与疾病相关联的基因功能区域,有助于了解甲基化调控基因表达的机制,为后续研究提供疾病关联基因的线索.目前检验甲基化差异区域的方法存在一定不足:一是缺乏定义差异甲基化区域的统一标准;二是检测结果存在横跨多个基因功能区域的现象,难以进行生物学解释.本文基于得分检验(Zscore test)提出了以基因功能区域为单位的检验差异甲基化水平的方法cZST.该方法的特点是依靠位点物理距离和染色体全局信息修正协方差矩阵.模拟研究表明cZST在亚硫酸氢盐测序数据样本量小、读数低、扰动位点多的情形下具有高功效,且优于现有方法.将cZST运用到乳腺导管癌真实数据中,检测出与疾病显著相关的661个基因功能区和81个单位点.基因注释分析说明了cZST方法的有效性,并发现Dbl、Pleckstrin基因家族及Rho调控基因的甲基化与乳腺导管癌紧密相关,为后续研究提供指引.     

Detection of DNA methylation changes during seed germination in rapeseed （Brassica napus）

DNA methylation is a common yet important modi- fication of DNA in eukaryotic organisms. DNA methy- lation, especially methylation of cytosine (m5C), have both epigenetic and mutagenic effects on various cellu- lar activities such as differential gene exp…