H2BC21

A research team has recently made a significant breakthrough in understanding how the DNA copying machine helps pass on epigenetic information to maintain gene traits at each cell division. Understanding how this coupled mechanism could lead to new treatments for cancer and other epigenetic diseases by targeting specific changes in gene activity. A research team led by Professor Yuanliang ZHAI at the School of Biological Sciences, The University of Hong Kong (HKU) collaborating with Professor Ning GAO and Professor Qing LI from Peking University (PKU), as well as Professor Bik-Kwoon TYE from Cornell University, has recently made a significant breakthrough in understanding how the DNA copying machine helps pass on epigenetic information to maintain gene traits at each cell division. Understanding how this coupled mechanism could lead to new treatments for cancer and other epigenetic diseases by targeting specific changes in gene activity. Their findings have recently been published in Nature. Background of the Research Our bodies are composed of many differentiated cell types. Genetic information is stored within our DNA which serves as a blueprint guiding the functions and development of our cells. However, not all parts of our DNA are active at all times. In fact, every cell type in our body contains the same DNA, but only specific portions are active, leading to distinct cellular functions. For example, identical twins share nearly identical genetic material but exhibit variations in physical characteristics, behaviours and disease susceptibility due to the influence of epigenetics. Epigenetics functions as a set of molecular switches that can turn genes on or off without altering the DNA sequence. These switches are influenced by various environmental factors, such as nutrition, stress, lifestyle, and environmental exposures. In our cells, DNA is organised into chromatin. The nucleosome forms a fundamental repeating unit of chromatin. Each nucleosome consists of approximately 147 base pairs of DNA wrapped around a histone octamer which is composed of two H2A-H2B dimers and one H3-H4 tetramer. During DNA replication, parental nucleosomes carrying the epigenetic tags, also known as histone modifications, are dismantled and recycled, ensuring the accurate transfer of epigenetic information to new cells during cell division. Errors in this process can alter the epigenetic landscape, gene expression and cell identity, with potential implications for cancer and ageing. Despite extensive research, the molecular mechanism by which epigenetic information is passed down through the DNA copying machine, called the replisome, remains unclear. This knowledge gap is primarily due to the absence of detailed structures that capture the replisome in action when transferring parental histones with epigenetic tags. Studying the process is challenging because of the fast-paced nature of chromatin replication, as it involves rapid disruption and restoration of nucleosomes to keep up with the swift DNA synthesis. In previous studies, the research team made significant progress in understanding the DNA copying mechanism, including determining the structures of various replication complexes. These findings laid a solid foundation for the current research on the dynamic process of chromatin duplication. Summary of Research Findings This time, the team achieved another breakthrough by successfully capturing a key snapshot of parental histone transfer at the replication fork. They purified endogenous replisome complexes from early-S-phase yeast cells on a large scale and utilised cryo-electron microscopy (cryo-EM) for visualisation. They found that a chaperone complex FACT (consisting of Spt16 and Pob3) interacts with parental histones at the front of the replisome during the replication process. Notably, they observed that Spt16, a component of FACT, captures the histones that have been completely stripped off the duplex DNA from the parental nucleosome. The evicted histones are preserved as a hexamer, with one H2A-H2B dimer missing. Another protein that involved in DNA replication, Mcm2, takes the place of the missing H2A-H2B dimer on the vacant site of the parental histones, placing the FACT-histone complex onto the front bumper of the replisome engine, called Tof1. This strategic positioning of histone hexamer on Tof1 by Mcm2 facilitates the subsequent transfer of parental histones to the newly synthesised DNA strands. These findings provide crucial insights into the mechanism that regulates parental histone recycling by the replisome to ensure the faithful propagation of epigenetic information at each cell division. This study, led by Professor Zhai, involved a collaborative effort that spanned nearly eight years, starting at HKUST and concluding at HKU. He expressed his excitement about the findings, 'It only took us less than four months from submission to Nature magazine to the acceptance of our manuscript. The results are incredibly beautiful. Our cryo-EM structures offer the first visual glimpse into how the DNA copying machine and FACT collaborate to transfer parental histone at the replication fork during DNA replication. This knowledge is crucial for elucidating how epigenetic information is faithfully maintained and passed on to subsequent generations. But, there is still much to learn. As we venture into uncharted territory, each new development in this field will represent a big step forward for the study of epigenetic inheritance.' The implications of this research extend beyond understanding epigenetic inheritance. Scientists can now explore gene expression regulation, development, and disease with greater depth. Moreover, this breakthrough opens up possibilities for targeted therapeutic interventions and innovative strategies to modulate epigenetic modifications for cancer treatment. As the scientific community delves deeper into the world of epigenetics, this study represents a major step towards unravelling the complexities of replication-coupled histone recycling.

引言组蛋白变体H2A.Z在启动子上发现并调节转录。ATP依赖性染色质重塑器SRCAP复合体(SRCAP-C)促进标准组蛋白H2A.Z-H2B二聚体被H2A–H2B取代。2024年2月8日，复旦大学徐彦辉及Wang Li共同通讯在Cell Discovery（IF 33.5）在线发表题为“Structural insights into histone exchange by human SRCAP complex”的研究论文，该研究确定了人类SRCAP-C与含H2A的核小体结合的结构。在真核细胞中，基因组DNA包裹在组蛋白八聚体周围，在基因组上产生排列的核小体。典型核小体包含四个组蛋白H2A、H2B、H3和H4的两个拷贝。为了实现功能调节，ATP依赖性染色质重塑复合体(重塑复合体)改变核小体的组成和沿DNA的位置。染色质重塑复合物根据其催化ATP酶的异同可分为4个家族：模仿开关(ISWI)、染色质结构域解旋酶DNA结合(CHD)、开关/sucrose不可发酵(SWI/ SNF)和肌醇营养不良80 (INO80)。其中两种重塑蛋白由单个亚基(CHD家族)或几个亚基(ISWI家族)组成。相比之下，SWI/SNF和INO80家族是多亚基复合物，除了超家族II解旋酶样运动亚基外，还含有肌动蛋白和肌动蛋白相关蛋白(ARPs)以及其他具有不同功能的重塑蛋白特异性亚基。人类染色质重塑子SNF2相关CPB激活蛋白(SRCAP)复合物(SRCAP-C)及其酵母对应物SWR1复合物(SWR-C)属于INO80染色质重塑子家族。SRCAP-C和SWR-C可以用H2A.Z-H2B二聚体以ATP依赖的方式取代H2A-H2B二聚体，这一过程可能需要组蛋白伴侣。生成的H2A.Z含核小体富集于几乎所有常染色质基因的启动子区域，并参与转录和其他过程的调控，如DNA复制、DNA修复和染色体分离。在真核细胞中，H2A.Z主要存在于它位于启动子的远端，在RNAPII的起始和延伸中起关键作用。H2A.Z在复制起点也广泛富集，并通过调节早期复制起点的起始和复制时间与DNA复制直接相关。此外，研究表明，协调H2A.Z双链断裂(DSBs)中的动力学对DNA修复至关重要。H2A.Z在染色体分离过程中也起着至关重要的作用，以维持基因组处于稳定状态。与其他染色质重塑子不同，SWR-C与核小体结合并诱导DNA展开和重新包裹，但不产生净DNA易位，表现出一种典型特征。核小体结合的SRCAP-C的整体结构（Credit: Cell Discovery）人SRCAP-C是一种~1-MDa复合物，由SRCAP、YL1、RUVBL1、RUVBL2、ARP6、ZNHIT1、DMAP1、ACTL6A、ACTB和YEATS4等10个亚基组成，酵母SRCAP-C由10个相同的亚基组成，分别为Swr1、Swc2、RvB1、RvB2、ARP6、Swc6、Swc4、Arp4、Actin、Yaf9和另外4个酵母特异性亚基Swc3、Swc5、Swc7和Bdf1。先前的研究已经揭示了酵母SWR-C、人SRCAP-C和核小体结合酵母SWR-C在ATP类似物ADP-BeFx存在下的结构。SWR-C含有RuvBL蛋白的异六聚体，Swr1中的插入物通过RuvBL六聚体延伸。Swr1的ATP酶结构域在超螺旋位置(SHL) 2上抓住DNA，导致DNA在ADP-BeFx结合状态下移位1bp。此外，进入DNA部分被解开，组蛋白核心“弯曲”。与酵母SWR-C相比，SRCAP-C采用了大致相似的结构，包含一个带有两个独立臂的环状“头”。该研究测定了核小体结合的SRCAP-C在不同核苷酸结合状态下的结构，并进行了结构引导染色质免疫沉淀测序(ChIP-seq)分析，以测试ZNHIT1对H2A.Z在体内掺入的影响。SRCAP-C介导的Z交换。SRCAP亚基集成了一个6亚基肌动蛋白相关蛋白(ARP)模块和一个含ATP酶的马达模块。ATP酶相关的ARP模块沿DNA包围核小体的一半，并可能抑制净DNA易位，这是SRCAP-C的独特特征。motor模块在载脂蛋白(无核苷酸)、ADP结合和ADP-BeFx结合状态下采用不同的核小体结合模式，表明ATP酶驱动的运动通过解开进入DNA的包裹使H2A-H2B不稳定，并通过ZNHIT1亚基将H2A-H2B拉出核小体。结构导向的染色质免疫沉淀测序分析证实了H2A接触ZNHIT1维持H2A.Z基因组上占用率的必要性。该研究为SRCAP-C介导的H2A-H2A.Z交换机制提供了结构性的见解。原文链接https://www-nature-com.libproxy1.nus.edu.sg/articles/s41421-023-00640-1责编|探索君排版|探索君文章来源|“iNature”End往期精选围观一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版）热文Science最新发布：全世界最前沿的125个科学问题热文Nature | 2024年值得关注的七项技术热文Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望热文Nature | 癌症与神经系统的致命舞蹈

真核生物异染色质是高度压缩的染色质区域，富集了DNA/组蛋白甲基化等多种转录沉默型表观修饰和特异的组蛋白变体等因子，对维持该区域的基因沉默和基因组稳定性起着至关重要的作用。染色质重塑蛋白可通过调节核小体上DNA和组蛋白八聚体之间的相互作用，移动、重排和重组染色质纤维，从而改变染色质的紧密程度和三维结构，影响基因的表达。R-loop是由RNA:DNA杂合链和单链DNA组成的三链核酸结构，在基因组上广泛存在。孙前文实验室前期的全基因组R-loop检测发现大量低峰度的R-loop富集于拟南芥基因组的异染色质区域1-3，但其相应的功能和生物学意义仍然未知。2023年8月11日，清华大学生命学院孙前文实验室在《科学·进展》（Science Advances）杂志上发表了题为“DDM1介导的R-loop解除和H2A.Z排斥促进拟南芥异染色质的形成”（DDM1-mediated R-loop resolution and H2A.Z exclusion facilitates heterochromatinformation in Arabidopsis）的研究论文4，揭示了拟南芥中染色质重塑蛋白DDM1可通过解除R-loop结构并排除组蛋白变体H2A.Z来启动异染色质形成的机制。该研究中，作者收集了众多影响拟南芥异染色质状态的突变体并进行了全基因组R-loop测序，结果发现染色质重塑蛋白DDM1的突变会引起着丝粒周围区域异染色质R-loop水平显著升高（图1）。图1  DDM1突变导致着丝粒附近R-loop水平升高DDM1是SWI/SNF家族蛋白的一员，早在1993年就发现其突变后会导致全基因组甲基化水平降低，但背后的机制仍然未知。通过ChIP-seq结果显示DDM1富集在染色体的着丝粒周围区域，而DDM1的突变会引起该区域的异染色质水平下降以及转录水平升高。有趣的是，转录抑制剂FLA可以显著降低ddm1突变体中R-loop在着丝粒附近的富集，同时部分回补DNA甲基化在ddm1中的缺失（图2）。这些结果说明，DDM1可抑制与转录耦联的R-loop异常积累，从而确保着丝粒附近的DNA甲基化等异染色质状态正常建立。图2  转录耦联R-loop破坏着丝粒附近异染色质DDM1的N端和C端都有保守的解旋酶结构域（图3A）,并且拟南芥ddm1-1突变体在C端解旋酶保守结构域中发生了C615Y的氨基酸残基突变。体外结果显示，RNA:DNA杂合链可以显著激活DDM1蛋白的ATPase活性，并且体外重组的DDM1蛋白可以RNA从RNA:DNA杂合链结构中解离，但是突变体蛋白DDM1C615Y的解旋酶活性显著下降（图3）。结合in vivo数据，表明DDM1可以直接清除在异染色质区域瞬时转录产生的R-loop结构，并促进异染色质形成。图3  DDM1解除RNA:DNA杂合链为了深入探索DDM1促进异染色质形成的机制，作者通过结合免疫沉淀-质谱分析寻找DDM1的互作蛋白。结果显示，DDM1可以和多种H2A组蛋白变体互作，其中H2A.W与H2A.Z两种变体在基因组上的分布是互斥的（图4A和B）。ChIP-seq结果显示，DDM1功能缺失的突变体中，着丝粒区域的H2A.W含量下降，而H2A.Z含量上升（图4C），说明野生型中异染色质区域定位的DDM1阻止了H2A.Z在该区域富集。体外互作结果表明，DDM1可以分别与H2A.W-H2B、H2A.Z-H2B二聚体互作（图4D），但是H2A.W-H2B可以抑制DDM1与H2A.Z-H2B复合物的形成（图4E），这也解释了在野生型中DDM1可以帮助H2A.W定位在着丝粒周围区域、同时抑制H2A.Z在该区域的富集这一现象。有趣的是，DDM1与H2A.W-H2B、H2A.Z-H2B均可抑制DDM1的解旋酶活性（图4F），而H2A.W的三突变体h2a.w-3中R-loop水平并没有显著升高（图4C），表明DDM1解除R-loop和调控H2A变体定位的这两种功能相互独立。图4  DDM1抑制H2A.Z在着丝粒附近富集由于H2A.Z在染色体上的定位由SWR1复合物介导，作者在ddm1背景下对拟南芥SWR1复合物的关键因子ARP6进行敲除，发现ddm1arp6双突可以一定程度上降低H2A.Z的积累，同时回补ddm1升高的R-loop和降低的DNA甲基化水平（图5）。这些结果说明，DDM1可以抑制H2A.Z在着丝粒周围区域的积累，同时解除由H2A.Z富集带来的转录耦联R-loop，从而促进异染色质的形成。图5  抑制H2A.Z的富集可回补ddm1中R-loop的异常积累和DNA甲基化缺失最后，作者发现DDM1抑制H2A.Z、解除R-loop的功能与花粉核的形态密切相关。野生型的花粉中，DDM1在精核（SN, sperm nuclei）中富集，而H2A.W也在精核中积累，但H2A.Z和R-loop在营养核（VN, vegetative cell nuclei）中富集（图6A-C）。DDM1功能缺失后，H2A.Z和R-loop信号可以扩散到精核中（图6B和C），但H2A.W在精核中的聚集状态没有发生改变（图6A）。同时，ddm1突变体中异常富集的H2A.Z与R-loop伴随着花粉精核变大、空洞的形成（图6D）。图6  DDM1在精核中解除R-loop和抑制H2A.Z富集综上所述，本项研究揭示了拟南芥的一种染色质重塑蛋白DDM1的主要功能是排斥异染色质区域的H2A.Z积累并解除R-loop，从而启动异染色质的形成，阐明DDM1维持基因组DNA甲基化的分子新机制和生物学新功能（图7）。图7  DDM1促进异染色质形成的工作模式图原文：https://www-science-org.libproxy1.nus.edu.sg/doi/10.1126/sciadv.adg2699会议推荐会议详情会议名称：2023（第14届）细胞与基因治疗研讨会暨抗体工程与创新免疫治疗技术论坛主办单位：生物谷、梅斯医学、同济大学附属同济医院、南京可缘大会时间：9月22-23日大会地点：上海 大会规模：1500人2010年至今，生物谷联合各合作单位已经顺利召开了13届细胞与基因治疗研讨会，与全球致力于细胞治疗行业同仁们一同成长沉淀，在提供行业信息交流平台的同时，也为基础研究提供了技术/产品等产业化的孵化平台。基于此，本届会议将继续以转化医学为切入点，以基础研究与临床应用相结合，针对细胞治疗的临床前沿研究、临床监管、治疗规范、细胞治疗安全性，免疫细胞新型疗法、实体瘤治疗、干细胞与癌症、抗体药物、核酸药物、基因编辑、肿瘤免疫治疗等热门议题进行讨论，诚邀国内外产学研医专家共聚，共绘产业发展新蓝图！长按识别二维码立即报名参会