For the first time, the high-resolution structure of de novo DNA methyl transferase and natural substrate nucleosomes was analyzed

The Xu Huaqiang Task Force of the Shanghai Drug Research Institute and the Peter Jones Task Force of the Wen'anlo Institute of the United States and the Karsten Melcher Task Force published the important research results entitled Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B online on September 23, 2020 in the international top journal NATURE. Using Cryo-EM technology, the team first analyzed the high-resolution structure of denovo DNA methyl transferase (DNMT3A2/DNMT3B3) and natural substrate nucleosomes, expounded the binding pattern of DNMT3A2/DNMT3B3 and nucleosomes, and proposed a model of methylation of whole genome DNA.
DNA methylation can change chromatin structure, DNA stability and DNA-protein interactions to control gene expression. DNA methylation can be passed on to newborn children's DNA along with the process of DNA replication, which is an important prescicational genetic mechanism. In a chromatin environment, dna methylation is much more complex than in solutions, and nucleosomes, as components of genetic material, are more difficult to methylate the DNA that wraps around their periphery. However, most nuclear gadgets bind to the denovo DNA methyl transfer enzyme inactive. DenovoDNA methylation metastases 3A and 3B catalyzed CpG methylation are essential for mammalian development and cell differentiation and are often closely related to the occurrence of cancer. By analyzing the expression of different subsypes of DNMT in a large number of normal tissues (GTEx databases) and cancer organizations (TCGA databases), the study focused on the interaction of DNMT3A2 and DNMT3B3 with the nucleus of the nucleus, as these are the two main DNMT subsypes in human cancer. The DNMT3A catalytic domain and the DNMT3L catalytic domain, as well as their crystal structure with free DNA, have been parsed. However, due to its limitations, the mechanism of interaction between DNMT and its natural substrate nucleosome has not been explained.The Xu Huaqiang Task Force of the Shanghai Drug Research Institute, in cooperation with the Peter Jones Group of the Wenanlo Institute of the United States and the Karsten Melcher Research Group, has long been committed to studying the important effects of DNA methylation on gene expression regulation and its extensive participation in the development of human cancer. In order to reveal the interaction between DNMT3A2/3B3 and nucleosomes and to understand DNA methylation on chromosomes, through more than four years of unremitting efforts, the frozen electron mirror technology was used to successfully analyze the frozen electron mirror structure of DNMT3A2/3B3 and the near-atomic resolution of nuclear small complexes. The structure shows that the heterogeneum tumoid complex (3B3-3A2-3A2-3B3) is very similar to the separated DNMT3A catalytic domain domain and the DNMT3L catalytic domain domain complex, but interacts symmetrically with the nuclear gadget. One of the catalytic domains of class DNMT3B3 is anchored in the acid patch (acidpatch) region of the nucleosome, and its core area of action is the DNMT3B3 740-bit and 743-bit arginine finger. The acidic patch area of the nucleosome interacts critically with a variety of nucleosome binding proteins. The DNMT3A2 catalytic domain does not interact with the core region of the nucleosome, but with the path of DNA, with the connected DNA (linkerDNA) at one end and catalysis its CPG methylation. Although the DNMT3 family protein is highly conservative, the structural comparison of the DNA-binding DNMT3A2 and the core region of the nucleosome reveals the switching function of the target identification region (TRD) domain. All DNMT3 subsypes with catalytic activity contain TRD domains that are critical to the action of target DNA, which spatially blocks the interaction of catalytic domains with the core regions of nuclear small bodies, but enhances the binding ability of DNA.Figure 1:a, the electron density map of the frozen electroscope of the complex structure; b, acid patch interaction; c, DNMT3A2 catalytic domain and connected DNA interaction and DNMT3B3 catalytic domain interaction with the nucleosome core.
In order to verify the importance of acid patch interactions for nucleosome collection, a mutation analysis of arginine fingers (R740 and R743) was performed and the amino acids (K745 and R749) were used as a control away from the acidic patch area. The INostophane Interaction Experiment (ALPHAScreen) showed that mutations in the opposite charge in the interaction core regions 740 and 743 arginine significantly reduced the interaction between the DNMT3A2/3B3 and the nucleosome, while mutations in the non-core region or the same charge did not change significantly in binding capacity as expected. Intracertic chromatin binding capability experiments have also been shown to interact with acidic patches 740 and 743 arginine finger mutations that lead to significant reductions in binding to chromatin. The DNA Methylation Array (Infinium Methylation EPIC BeadChip) also confirms the importance of the interaction between DNMT3B3 and nucleosome acid patches for DNA methylation reconstruction in the body. The mutant DNMT3B3 is closely related to the ability to restore DNA methylation and its binding to chromatin. As a control of the K745 and R749 mutations, methylation recovery levels were almost the same as 3B3 in wild DNMT3B3. In contrast, the R740E and R743E mutations that significantly reduce the binding of chromatin are much less efficient in methylation recovery. Limited microcosmicr nuclease digestion experiments (MNasedigestion) further confirmed the presence of the DNMT complex, either side of the nucleus significantly increased the protective area of about 10 base pairs. These data strongly support the importance of the interaction between the catalytic domain of the DNMT complex and acidic patches for nuclear small body collection and DNA methylation, and the binding to DNA does not depend on the active bit of the enzyme CpG.2: A AlphaScreen experiment determines the interaction between the His-labeled DNMT3A2-DNMT3B3 complex and the biotin-labeled NCP. b.DNMT3 chromosome binding experiment. c.HCT116 cells and DKO8 co-transmerate methylated heat maps of CpG bits of DNMT3B3 cells. d. The box chart shows the horizontal distribution of DNA methylation of 109,998 probes.
In summary, the structure and functional analysis of DNMT3A2/3B3 and the nuclear small complex reveal that the DNMT3B3 catalytic structure domain has unexpected nuclear small body targeting function, and the DNMT3A2/3B3 catalytic domain is located in the nuclear small body connected DNA region, which is very important for the whole genome DNA methylation. By separating the DNMT core nuclear small body target and CpG methylation through the DNMT class catalytic domain and the catalytic domain, the DNMT3 complex can be raised near the nuclear small body with difficulty approaching, and the CpG methylation targeting is connected to the DNA region. This suggests that the transmission of DNA methylation into the DNA of the nucleosome in the body requires the remodeling of the nucleosome, for example through DNA replication, transcription, or other nuclear small body remodeling events.
The work was carried out in 2016 under the guidance of the drug institute's mentors, Xu Huaqiang and Winan Lo Peter Jones and Karsten Melcher, and is the only author of this paper, under the guidance of Xu Tinghai, a 2017 Ph.D. graduate of the Drug Institute and currently a co-postdoctoral student in the Peter Jones Group of the Winnenlo Institute in the United States. Dr. Minmin Liu of the Winanlo Institute, Dr. X. Edward Zhou, Dr. Gongpu Zhao and Professor Gangning Liang of the University of Southern California were also involved in the work. Xu
, of the Shanghai Drug Institute in China, and Peter Jones of the Wen'anlo Institute in the United States and Karsten Melcher in Shanghai, China, co-authors of this article. (Source: Science
.)