Reviews ZhangZhiguo(Columbia University).Li Guohong(Wuhan University).
Multicellular organisms, such as humans, have hundreds of cell types with different functions, and together these cells build a complete life organism. All of these diverse cell types originate from the same zygote, evolved through the process of cell** and cell differentiation, and carry the same DNA genetic information, and their differences in gene expression profile, cell traits and function are mainly due to epigenetic information. Epigenetic information is like a palette of life that determines the unique identity of a cell. Chromatin is a common vector of DNA genetic information and epigenetic information. Its basic building block is the nucleosome, which consists of 147 bp of DNA tightly wound around a histone octamer and contains one (H3-H4)2 tetramer and two H2A-H2B dimers. Nucleosomes are further organized and folded to form the higher-order structure of chromatin. Nucleosomes carry a variety of histone posttranslational modifications (PTMS) and are key components of epigenetic marks。These markers are elastically transmitted from parent cells to daughter cells at the time of cell**, which not only maintains epigenetic memory, but also provides the necessary window for cell differentiation to rewrite epigenetic information. This mechanism of epigenetic marker transmission is achieved through the deassembly of parental nucleosomes and the reassembly of nascent strand nucleosomes during DNA replication。As the DNA replication fork advances, it first deassembles the anterior parental nucleosomes so that the replicators can come into contact with the DNA, which in turn opens the double-stranded DNA (dsDNA) for DNA replication. In this process, histone chaperones and replicom constituent factors** dissociate the parental histones carrying epigenetic markers and redistribute them to two nascent daughter strand DNA to complete the assembly of nascent nucleosomes, thereby enabling the transmission of epigenetic information。If the epigenetic information transmission is disrupted during this process, it may lead to changes in chromatin structure and gene expression profile, which may further lead to cell type mutations, and may even lead to the occurrence of diseases such as cancer and aging, or metabolic abnormalities in the human body. The molecular machinery of DNA replication ---Replicas(replisome), the core of which is the replication helicase CMG, which is composed of MCM2-7 hexamer, CDC45 and GINS. In addition, numerous other replication factors are directly or indirectly assembled on the replicat, including lead-chain polymerase POL, lagging chain polymerase POL δ, primase POL, CTF4, etc. In addition, the Fork Protection Complex (FPC) and nucleosome chaperones are essential factors for DNA replication in the chromatin environment, ensuring the normal progress of DNA replication. In DNA replication-coupled nucleosome metabolism, FACT not only plays a role in the presentation of newly synthesized histones；It is also involved in the process of nucleosome disassembly and parental histone at the front end of the replication fork, but the molecular mechanism is unknown. Previous studies have shown that multiple replicatosome factors have histone chaperone activity and are involved in the transport and assignment of histones to nascent chains. For example, the N-terminal extension (NTE) of the MCM2 subunit contains a conserved histone binding domain (HBD) that can wind histone (H3-H4)2 tetramers in a way that mimics DNA binding and facilitate their assignment to nascent DNA lag strands。Although histone chaperones and replicator factors are already known to be involved in parental histone proteins**, the molecular mechanisms by which they facilitate the deassembly of parental nucleosomes coupled to DNA replication and subsequent parental histone delivery to nascent DNA strands during chromatin replication remain unclear. Currently, the structural study of replicator-synergistic histone complexes is very lacking, which greatly limits our comprehensive understanding of the mechanisms of chromatin replication. 6 March 2024, University of Hong KongZhai YuanliangProfessor, Peking UniversityGao NingProfessor, Peking UniversityLi QingProfessor, Cornell UniversityDai Bi XuanProfessors cooperate, innatureThe magazine published a publication entitled ".parental histone transfer caught at the replication fork"Research**. In this work, we captured the fact-histones-replisome complex, a key intermediate complex in the process of nucleosome disassembly and histone ** coupled to DNA replication, and analyzed the structure and mechanism to elucidate the key molecular mechanism of parental histone protein**. 
The work began in 2015, and the researchers successfully purified endogenous replicator complexes from large-scale cultured Saccharomyces cerevisiae cells by integrating macromolecular complex purification techniques such as affinity purification tags, cell cycle synchronization, chromatin enrichment, and gradient cross-linking, as well as extensive condition optimization. After a fine cryo-EM structural analysis, a replica complex bound to FACT and parental histones was captured and its 35 angstrom high-resolution cryo-EM structure (Figure 1). Consistent with the structural data, the quantitative mass spectrometry results showed that Histones, FACT and similar to other replica components had high abundances. 
Figure 1, cryo-EM structure of the replisome-fact-histones complex (a-c). In this replicat structure, histones exist as hexamers containing 1 (H3-H4)2 tetramer and 1 H2A-H2B dimer. The parental DNA is completely stripped from the histone, and the other H2A-H2B dimer has also been dissociated (Figure 2). A segment of the Helix-Turn-Helix of TOF1 is inserted between the parental DSDNA and histone hexamers, ensuring complete stripping of the DNA. FACT utilizes the intermediate domain (MD) and carboxy-terminal domain (CTD) of SPT16, which, together with MCM2-HBD, occupies the binding surface exposed upon dissociation of parental DNA and H2A-H2b dimers on histone hexamers (Figure 2). Here, a long helix segment of MCM2-HBD occupies the binding interface of dissociated H2A-H2B, while a loop region is wound around the exposed DNA-binding interface on one of the H3-H4 dimers (Figs. 2D, 2F). Interestingly, the DNA binding interface of the other H3-H4 dimer was fully exposed, which may leave room for binding of downstream factors in the histone ** pathway (Figure 2D). A loop region in the tail of SPT16-CTD binds to the DNA binding interface exposed by the H2A-H2B dimer, facilitating the binding of the H2A-H2B dimer to the (H3-H4)2 tetramer (Figs. 2C, 2E). To sum up,SPT16, MCM2-HBD, and TOF1-HTH protect the binding interface exposed by histone hexamer dissociation of DNA in the form of chaperones, thereby maintaining the stability of histone subcomplexes after nucleosome disassembly. 
Figure 2, histone hexamers and their chaperones in replicator replications. (a-b) histone hexamers; (c-d) binding to histone-chaperone complexes in replicades; (e-f) nucleosomes. At the front end of the replication fork, MCM2-NTE and TOF1 interact closely to form a binding platform for histone hexamers (Figure 1), bringing the hexamer close to the CTF4 region, which happens to be the binding site of lagging strand DNA polymerases. Consistent with this structural observation, it has been reported that MCM2-HBD promotes parental histone assignment to the lagging strand of nascent DNA。Therefore, the interaction between MCM2-NTE and TOF1 may affect the partitioning of parental histones between the leader and lagging chains to a certain extent. **Corresponding mutant strains were further designed to interfere with the binding of MCM2-NTE and TOF1. Espan (Enrichment and Sequencing of Protein-Associated Nascent DNA) analysis based on nascent DNA sequencing showed that disruption of the interaction between MCM2-NTE and TOF1 severely affected the proportion of parental histone** and newly generated lagging strand DNA. This work clearly shows the molecular details of a key intermediate step in the process of DNA replication coupling of parental histone **, elucidating the molecular mechanisms of FACT, TOF1, MCM2 and other in the process of parental histone **, indicating that histone hexamer is likely to be a kind of ** unit of parental histone, which lays an important foundation for comprehensively revealing the inheritance mechanism of epigenetic information of DNA replication coupling. It is worth mentioningscience china life scienceThe letter column of the journal was published in the Institute of Biophysics, Chinese Academy of SciencesXu RuimingThe research group is entitled ".structure of a histone hexamer bound by the chaperone domains of spt16 and mcm2The article published the crystal structure of the complex of the human FACT complex subunits SPT16, MCM2 and histones. The structure indicatesThe repurposed unit of the old nucleosome should be a histone hexamer composed of a (H3-H4)2 tetramer and an H2A-H2b dimer. (For the summary and comparison of the above two works, please refer to the special article review of Researcher Zhu Bing, which bioart pushes separately today) Professor Zhai Yuanliang, Professor Gao Ning, Professor Li Qing, and Professor Dai Bijun are the co-corresponding authors of this paper, and Prof. Li Ningning of Peking University, Gao Yuan, a PhD student of the University of Hong Kong, Zhang Yujie, a PhD student of Peking University, and Dr. Yu Daqi of the University of Hong Kong (formerly of the Hong Kong University of Science and Technology) are the co-first authors of this paper. Prof. Xiang Li, Prof. Yang Liu, Prof. Da Zhou, Dr. Jianwei Lin, Ph.D. students Jian Li and Dr. Zhichun Xu from the University of Hong Kong, Prof. Shangyu Dang and Dr. Yingyi Zhang from the Hong Kong University of Science and Technology, and Associate Researcher Jianxun Feng from Peking University also participated in this work. Zhai Yuanliang's research group at the University of Hong Kong is recruiting outstanding postdoctoral fellows and doctoral students for a long time, if you are interested, please refer to Bioart Recruitment: Zhai Yuanliang Research Group Recruitment, School of Biological Sciences, The University of Hong Kong, or visit the research group website: Li Qing's research group at Peking University, long-term recruitment of outstanding postdoctoral fellows and doctoral students, interested parties please refer to the research group webpage of the School of Life Sciences of Peking University: expert comments
Zhang ZhiguoThe stability and function of eukaryotic genomic DNA is strongly influenced by the structure and organization of chromatin. In the cellular** process, it is not only necessary to ensure accurate replication of the DNA of the entire genome, but also to reproduce the structural map of chromatin on the newly generated DNA strand. How to efficiently transfer and maintain different chromatin states during DNA replication is a core topic in epigenetics. Histones are the backbone proteins of nucleosomes, the basic unit of chromatin in eukaryotic cells. Parental histones carrying specific modifications are the most important vectors of epigenetic information. When DNA is replicated, the parental nucleosomes in front of each replication fork are rapidly disassembled to facilitate efficient DNA synthesis. Once replication is complete, histones reconstruct nucleosomes on the nascent DNA strands in two ways: from the newly synthesized histone assembly de novo, and by remodeling nucleosomes from parental histones. Nucleosome assembly is a gradual process in which H3-H4 tetramers are assembled first and then H2A-H2b dimers are rapidly recruited. In yeast cells, nascent H3-H4 dimers are guided by ASF1, and lysine at position 56 of H3 is acetylated by RTT109 (H3K56AC), after which ASF1 passes the H3-H4 dimer to downstream chaperones, including CAF-1 complexes, to assemble nascent (H3-H4)2 tetramers and form nucleosomes. However, our current understanding of how parental histones are effectively used to reconstruct relevant epigenetic information is still limited. Recent studies have found that some replicomal components have histone chaperone activity and are able to facilitate the transfer of parental H3-H4 to the newly synthesized DNA strand. In the case of budding yeast, analysis of the EPAN (protein-associated nascent DNA enrichment and sequencing) method showed that the two subunits of Pol, DPB3 and DPB4, were able to facilitate the transfer of parental histones to the leader strand DNA by interacting with H3-H4. In addition, MCM2 is an important subunit of MCM helicase with an N-terminus containing a conserved histone binding sequence (HBM). ESAN analysis also showed that the MCM2-HBM mutant with defective histone binding significantly affected the transfer of parental histones to lagging chains in yeast. The same results have been reported in mammalian cells such as mouse embryonic stem cells. These findings all confirm the conserved role of MCM2 in parental histone transfer. Although these breakthrough discoveries shed light on some aspects of the mechanism by which epigenetic information is transmitted during replication, the specific molecular mechanism by which parental histones are transferred by replicators remains a mystery. The work of the team of Prof. Zhai Yuanliang of the University of Hong Kong, Prof. Gao Ning and Prof. Li Qing of Peking University and Prof. Dai Bijun of Cornell University in the journal Nature has revealed for the first time the high-resolution frozen structure of parental histones during replication fork transfer, which is a milestone for understanding the genetic mechanism of histone-encoded epigenetic information. In this study, the research team successfully resolved the structure of endogenous replicators purified from yeast cells using cryo-EM technology. Excitingly, structural information shows that FACT and MCM2-HBD are able to bind to histone hexamers consisting of H3-H4 tetramers and one H2A-H2B dimer. This histone hexamer is completely separated from the parental DNA molecule and temporarily transferred to the surface of the TOF1 protein, which is located at the front end of the replicator. More importantly, the research team also discovered the interaction between MCM2-HBD and TOF1, which mediates the transfer of parental histones to lagging chains. These findings provide valuable clues and directions for in-depth study of the transmission and mechanism of parental histones during replication. However, there are still many issues that need to be addressed urgently. For example, how histone octamers form (H3 H4)2-H2A-H2B dimers and one H2A H2B dimer during parental nucleosome breakdown, and whether this histone hexamer unit is utilized as a whole still needs further research. In addition, the fate of the dissociated parental H2A-H2B is also a perplexing issue. At the same time, how parental histones are efficiently transferred to the leading strand DNA, and the role that pol plays in this process, also needs to be revealed. The structural information revealed by this work provides a solid foundation for in vitro reconstruction of the disassembly and subsequent reassembly of replicat-coupled nucleosomes, as well as related functional analysis. With the deepening of research, we have reason to believe that in the future, more intermediates** of parental histone transfer at replication forks will be obtained, which will help us to more clearly reveal the exact role of histones in the newly synthesized leader and lagging chains. Research in this area will provide us with a new perspective to examine and understand the mechanisms of transmission and maintenance of epigenetic information during DNA replication. Expert commentary
Li GuohongOn March 6, Professor Zhai Yuanliang of the University of Hong Kong, Professor Gao Ning and Professor Li Qing of Peking University, and Professor Dai Bijun of Cornell University published a study entitled "Parental Histone Transfer Caught at the Replication Fork" in the journal Nature**. In this study, we successfully captured the high-resolution cryo-EM structure of a key intermediate state of the DNA-replisome-fact-histone complex during DNA replication using yeast-purified endogenous replica samples, opening up a new perspective for understanding the DNA replication process based on chromatin structure. The genomic DNA of eukaryotes is always stored inside the nucleus as the formation of chromatin. In the process of cellularity, the replication inheritance of chromatin state is a key core issue in maintaining cell fate. In the process of DNA replication, nucleosomes, as the basic units of chromatin structure, accompany the DNA replication fork for dynamic deassembly and reassembly. The structure of the parental nucleosomes before the replication fork needs to be disassembled, and the DNA is exposed to facilitate the replication fork to open the DNA duplex for DNA replication. At the same time, the histones on the parental nucleosomes are reassembled to the newly generated daughter strand DNA behind the replication fork to form nucleosomes, so as to realize the transmission and inheritance of epigenetic information. The two most central events in the inheritance and maintenance of epigenetic information on histones are: 1) how the parental histones are randomly and evenly distributed to the newly synthesized daughter strands, the leading and lagging strands, to form nucleosomes during DNA replication; and 2) how newly synthesized histones are assembled to form nucleosomes and reconstruct epigenetic information to ensure accurate transmission of epigenetic information between parenting and generation. Zhu Bing's team in China found that during DNA replication, histone H3-H4 tetramers were assigned to the newly synthesized daughter strand DNA in a fully retained manner, revealing the molecular mechanism of epigenetic modification inheritance on histone H3-H4. Zhiguo Zhang's team at Columbia University and Anja Groth from the University of Copenhagen accurately measured the relative distribution of histones and their modifications on the leader and lagging chains, and found that the vast majority of histone modifications showed a symmetrical distribution between the two new synthesizer chains. Surprisingly, however, Zhang's team recently discovered that H3K9Me3 exhibits a very unique distribution pattern, which is significantly enriched on the LINE transposition element of the leading chain. Using single-molecule technology, our team and collaborators have discovered that histone chaperone FACT has dual functions of nucleosome deassembly and reassembly, and plays an important role in nucleosome deassembly and parental histone ** during DNA replication and gene transcription. In summary, a variety of protein factors have been found to be involved in this process, but it is still unclear how these factors promote DNA replication-coupled deassembly of parental nucleosomes and parental histones** and their reassembly on nascent nucleosomes. In this research work, the collaborative team successfully purified endogenous replica complexes from large-scale cultured cell cycle-synchronized Saccharomyces cerevisiae cells for the first time by using macromolecular complex purification techniques such as affinity purification tags, chromatin enrichment, and gradient cross-linking, and successfully obtained replicasome complexes with fact and parental histone proteins35 angstrom high-resolution cryo-EM structure. The structure clearly shows the molecular details of a key node in the process of DNA replication coupling of parental histone **, revealing the process of histone detachment from parental DNA in the form of hexamers in the form of spt16, MCM2-HBD and TOF1-HTH before replication fork, and combined with nascent DNA sequencing ESAN experiments, the molecular role of FACT and chromatin replication factors such as TOF1 and MCM2 in histone ** and delivery to the nascent lagging strand is clarified. This study lays an important foundation for comprehensively elucidating the transmission and inheritance mechanism of epigenetic information coupled with DNA replication. Interestingly, this study results found for the first time that the parental histones may be delivered in the form of hexamers, including one (H3-H4)2 tetramer and one H2A-H2B dimer during DNA replication, which not only supports the partitioning pattern of H3-H4 tetramer full retention found in the previous period, but also provides support for the genetic mechanism of the epigenetic modification information on **H2A-H2B in the chromatin replication process. Early studies were based on the high dynamics of H2A-H2B dimers on chromatin, and the inheritance of histone epigraphers was mostly studied based on H3-H4 tetramers in chromatin replication studies, while the inheritance of epigenetic information on H2A-H2B was neglected. Our team has previously found that 30 nm chromatin fibers can promote the rapid spread and reconstruction of H3K27Me3 and H2AK119UB1 during DNA replication through the nucleosome-nucleosome pairing principle. Recently, Anja Groth's team at the University of Copenhagen found that the epigenetic information on the parental histones H2A-H2B during DNA replication can also be rapid** and reconstructed before H3-H4, thus enabling rapid epigenetic memory, supporting our view that H2AK119UB1 is epigenetic memory. At the same time, this work also provides a new perspective on the role of FACT complexes in chromatin replication. As histone chaperones, FACT complexes are involved in maintaining nucleosome integrity and regulating the assembly and deassembly of nucleosome structures, and play an important role in all chromatin-related biological processes, including transcription, DNA replication, and damage repair. Early studies have found that FACT is involved in the deassembly of pre-replication nucleosomes and plays a role in the presentation of parental histones** and newly synthesized histones, but the specific mechanism has not been clear. This study revealed for the first time the molecular basis of the coupling of FACT complexes to replicators and synergistically promoted the replication fork of parental histone hexamers**, and systematically compared the changes of FACT complexes from binding nucleosomes to subnucleosomes to histone hexamers and histone H3-H4 tetramers, laying a foundation for understanding the molecular mechanism of dynamic regulation of nucleosomes by FACT complexes. However, we have recently found that the nucleosome disassembly and reassembly functions of the FACT complex are regulated by a variety of epifactors, such as histone H2AK119 and H2BK120 ubiquitin modifications, and histone variant H2AZ, H2AX, MacroH2A, etc. It remains to be seen whether these different apparent regulatory messages interact with the FACT complex during chromatin replication, regulate the corresponding parental histones**, or are delivered to the nascent posterior hysteresis or leading strands**. In addition, as a histone octamer on the parental nucleosome, the fate of another parental H2a-H2B dimer is also worthy of further study. After it is dissociated from histone octamers by a long section of HCM2-HBD, it is maintained in this structure by binding to other components of the complex, but the high dynamics cannot be resolved, and it remains to be seen whether it is completely separated. According to the review, the experimental design of this work of the cooperative team is ingenious, the experimental system is complex, the difficulty is large, the content is rich, the research ideas are novel, and the structure is very beautiful. This study not only opens a new perspective for understanding the DNA replication process based on chromatin structure, but also clarifies the important functions of chromatin replication factors such as FACT, TOF1 and MCM2 in parental histone protein ** and delivery, and lays an important foundation for comprehensively elucidating the transmission and inheritance mechanism of epigenetic information during DNA replication. Here, I would like to congratulate the collaborating team again for making another important progress in the research direction of DNA replication regulation mechanism, and look forward to more breakthroughs in follow-up research. Original link:
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