On how mammalian transcription factors recognize methylated DNA

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.     

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD–DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16^INK4a genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions

We developed a method for site-selective CpG methylation of the budding yeast genome. The method recruits LexA-fused M.SssI DNA methyltransferase to LexA operator sequences integrated adjacent to the target site. Microarray analysis of methylated DNAs indicated that the tethered enzyme selectively methylates the region around the target site. Exploiting this method to methylate bait DNA in the one-hybrid system, we demonstrated methylation-dependent DNA binding of methyl-CpG binding proteins, MBD1 and Kaiso, in vivo. This methylation-dependent one-hybrid system would provide a versatile tool for the search and analysis of proteins that recognize methylated DNA to participate in epigenetic regulation.     

Characterization of Arabidopsis thaliana methyl-CpG-binding domain (MBD) proteins

Cytosine methylation at symmetrical CpG and CpNpG sequences plays a key role in the epigenetic control of plant growth and development; yet, the way by which the methylation signal is interpreted into a functional state has not been elucidated. In animals, the methylation signal is recognized by methyl-CpG-binding domain (MBD) proteins that specifically bind methylated CpG dinucleotides. In Arabidopsis thaliana, 12 putative MBD proteins were identified and classified into seven subclasses. Here, we characterized six MBD proteins representing four subclasses (II, III, IV, and VI) of the Arabidopsis MBD family. We found that AtMBD7 (subclass VI), a unique protein containing a double MBD motif, as well as AtMBD5 and AtMBD6 (subclass IV), bind specifically symmetrically methylated CpG sites. The MBD motif derived from AtMBD6, but not from AtMBD2, was sufficient for binding methylated CpG dinucleotides. AtMBD6 precipitated histone deacetylase (HDAC) activity from the leaf nuclear extract. The examined AtMBD proteins neither bound methylated CpNpG sequences nor did they display DNA demethylase activity. Our results suggest that AtMBD5, AtMBD6, and AtMBD7 are likely to function in Arabidopsis plants as mediators of the CpG methylation, linking DNA methylation-induced gene silencing with histone deacetylation.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

鲫Kaiso基因cDNA的克隆和表达分析

在爪蟾和斑马鱼中, Kaiso是一种在整个基因组范围内与甲基化CpG序列特异性结合的转录抑制因子, 在调控被甲基化基因表达的时间模式中起重要的作用。为深入研究DNA甲基化对我国重要养殖鱼类生殖和发育的影响, 我们克隆了鲫Kaiso基因的cDNA序列, 并对其时空表达模式进行了分析。该cDNA全长3145 bp, 5′-非翻译区132 bp, 3′-非翻译区1117 bp, 开放阅读框1896 bp, 编码631个氨基酸。鲫Kaiso蛋白与其他物种Kaiso蛋白的同源性分析表明, 与其他物种一样, 其 N端和C端分别有高保守性的BTB/POZ结构域和锌指结构域。整胚原位杂交结果显示, Kaiso mRNA在早期胚胎发育的各个时期均广泛表达, 信号均一, 但从尾芽期开始出现组织特异性表达差异。对不同发育阶段胚胎的实时定量PCR检测结果表明: 卵子中有高丰度的母源Kaiso mRNA存在; 在卵裂期至囊胚中期胚胎中Kaiso mRNA的丰度逐渐降低; 从囊胚中期至原肠早期都维持在最低水平状态; 原肠后期其表达水平又逐渐升高, 至尾芽期达到与未受精卵中相当的高水平后在器官发生期的整体水平又稍有下降。Kaiso mRNA丰度在胚胎发育早期的这种变化过程提示在卵裂期检测到的mRNA可能都是母源mRNA, 合子核Kaiso基因可能是在囊胚晚期后才开始转录。对成体不同组织的实时定量PCR检测结果表明Kaiso的表达存在明显的组织特异性差异, 在鲫肌肉、视网膜、心脏和脑中表达水平较高, 而在肾、胰、肝等器官中表达水平很低。Kaiso表达的时间和组织特异性提示其作为甲基化基因的转录抑制因子参与了胚胎和成体基因表达时空模式的调控。这些结果为进一步研究Kaiso和DNA甲基化修饰在鲫发育调控和遗传育种中的作用提供了基础资料。     

Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.     

No Evidence for AID/MBD4-Coupled DNA Demethylation in Zebrafish Embryos

The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos.     

Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2.

MeCP2 is a chromosomal protein which binds to DNA that is methylated at CpG. In situ immunofluorescence in mouse cells has shown that the protein is most concentrated in pericentromeric heterochromatin, suggesting that MeCP2 may play a role in the formation of inert chromatin. Here we have isolated a minimal methyl-CpG binding domain (MBD) from MeCP2. MBD is 85 amino acids in length, and binds exclusively to DNA that contains one or more symmetrically methylated CpGs. MBD has negligable non-specific affinity for DNA, confirming that non-specific and methyl-CpG specific binding domains of MeCP2 are distinct. In vitro footprinting indicates that MBD binding can protect a 12 nucleotide region surrounding a methyl-CpG pair, with an approximate dissociation constant of 10(-9) M.