New publishment in Genome Biology 在GB新发表文章一篇

Link to the research article: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-021-02455-3Phase separation is widespread within cells, where thousands of proteins or nucleic acid molecules form a membrane-less "compartments" inside living cells, like oil droplets in water, and participate in multiple cellular processes without interfering with each other. 1,6-hexydiol (1,6-HD) is the only tool capable of dissolving phase-separated droplets simultaneously, so it has great potential to be applied to study the relationship between phase separation and other biological processes or structures at global-level. However, the conditions of 1,6-HD vary considerably between studies and may even trigger apoptosis, abnormal protein aggregation, chromatin "frozen" and other side effects, which greatly hinder the application of this tool. On the other hand, eukaryotic chromatin folds in the nucleus in a specific form, forming ordered three-dimensional structures in space, which are related to cell fate decision and disease occurrence. Recently, phase separation has been proposed to have a prominent role in 3D chromatin organization. However, there is only indirect or case evidence, and lack of global-level studies. On 17 August 2021, the study of Professor Ding Junjun’s team entitled “Time-dependent effect of 1,6-hexanediol on biomolecular condensates and 3D chromatin organization” is published in Genome Biology. For the first time, the using condition of 1,6-HD (1.5%, 2 min) with fewer side effects was optimized and tested on a variety of cell lines, and a time-resolved map of 3D chromatin structure upon1,6-HD treatment was provided to explore the relationship between phase separation and 3D chromatin organization. Time-dependent effect of 1,6-HD on phase-separated droplets and 3D chromatin organization In this study, the researchers first analyzed the effects of different concentrations and treatment durations of 1,6-HD and found that short-term exposure to 1.5% 1,6-HD dissolved phase-separated droplets whereas long-term exposure caused aberrant aggregation without affecting cell viability. Based on this condition, the researchers drew a time-resolved map of 3D chromatin organization and found that short-term treatment with 1.5% 1,6-HD resulted in reduced long-range interactions, strengthened compartmentalization, homogenized A-A interactions, B-to-A compartment switch and TAD reorganization, whereas longer exposure had the opposite effects. Furthermore, the long-range interactions between phase-component-enriched regions were markedly weakened following 1,6-HD treatment. In all, the study finds a proper 1,6-HD condition (1.5%, 2 min) to dissolve phase-separated droplets with limited side effects and provides a time-resolved map of chromatin organization during 1,6-HD treatment. The study demonstrate that 1,6-HD treatment has a time-dependent effects on phase-separated droplets and chromatin organization at different hierarchies. This time-resolved map would serve as a resource for exploring the role of phase separation in 3D chromatin organization. The research was co-authored by Xinyi Liu, Ph. D Candidate and Shaoshuai Jiang, Postdoc,  Zhongshan School of Medicine. Ding Junjun, Professor of Zhongshan School of Medicine at Sun Yat-sen University, is the sole corresponding author.细胞内广泛存在相分离(Phase Separation)现象,成千上万的蛋白或核酸分子在复杂的细胞内部形成一个一个无膜“隔间”,就像油滴在水中一样,彼此互不干扰地参与细胞内如转录调控、应激、蛋白质质量控制、DNA复制等多种重要的生物学过程【1, 2】。1,6-己二醇(1,6-HD)是一种能够使得相分离液滴溶解的化学小分子,它是目前唯一的能同时破坏多种相的工具,所以非常有潜力应用于研究全局层面上相分离现象与其他生物学进程或结构之间的关系【3】。但是,在不同的研究中,1,6-HD的应用条件千差万别,没有统一的标准,某些条件甚至会导致细胞凋亡、蛋白异常凝集、细胞皱缩、染色质“冻结”等副作用,这大大阻碍了这一工具的应用【3-5】。 另一方面,真核细胞的染色质会以特定的形式折叠在细胞核内,在空间上形成有序的三维结构,这些三维结构对细胞生命活动至关重要,与细胞命运决定和疾病发生都有关系【6, 7】。那么染色质三维结构是如何被有序组织起来的呢?一直有假说认为相分离在其中发挥了功能,但目前只有间接的或点例的证据,缺乏全局水平上研究。 2021年8月17日,中山大学丁俊军课题组在Genome Biology 发表了题为Time-dependent effect of 1,6-hexanediol on biomolecular condensates and 3D chromatin organization的文章,首次系统测试并报道了一个适用于多种细胞系、副作用较小的1,6-HD应用条件(1.5%, 2 min),并绘制了1,6-HD处理下动态的染色质三维结构图谱,用于探究相分离和染色质三维结构的关系。在该项工作中,研究人员首先在小鼠胚胎干细胞(mESCs)中测试了不同浓度和时间的1,6-HD处理对多种相分离液滴、细胞活力、细胞核体积和染色质动态性的影响。文章发现,1.5%的1,6-HD对相分离液滴具有时间依赖效应,短时间处理使得相分离液滴溶解,而长时间处理则使得其重建(图一),另外更高浓度(5%和10%)的1,6-HD在短时间处理(2 min)时已经会导致严重的细胞凋亡。因此,该研究首次系统测试了1,6-HD的条件并报道了低浓度、短时间(1.5%, 2 min)是优化的1,6-HD应用条件,这一条件在多种细胞系中都能在不导致细胞凋亡、不影响细胞核体积和染色质动态性的情况下破坏相分离,可以应用于研究相分离与其他生物学进程之间的关系。 研究人员进而绘制了低浓度1,6-HD处理下时间动态的染色质三维结构图谱,首次描述了相分离溶解和重建过程中染色质结构的变化。研究结果表明,短时间1,6-HD处理会导致长距离互作减弱、短距离互作增加、基因组区室化增强、激活的基因组区室(Compartment A)内部互作更加均匀、抑制的基因组区室(Compartment B)向Compartment A转变、拓扑相关结构域(TAD)重组,而长时间的1,6-HD处理则具有相反的影响(图二),这些结果说明1,6-HD对不同层级的染色质三维结构的影响也具有时间依赖效应,这将相分离与染色质三维结构联系起来。除了分析1,6-HD对整体的染色质结构的影响,该研究还结合ChIP-seq数据鉴定了相分离组分富集的染色质相互作用。文章发现这些相互作用都是较长距离的,而且在1,6-HD处理之后剧烈减弱了(图三)。这些结果支持了一个新的模型,即相分离是通过维持长距离的染色质相互作用来维持不同层级的染色质三维结构的。总之,该项研究提供了一个优化的1,6-HD应用条件,有助于未来相分离的功能研究。基于这个条件,研究探讨了相分离与染色质三维结构的关系。该项研究优化了有力的研究工具,丰富了我们对染色质结构的认识,也为未来进一步研究特定的相分离液滴对染色质结构的具体影响提供了资源。 丁俊军课题组的博士研究生刘心仪和姜少帅博士为本文共同第一作者。丁俊军教授为这篇文章的唯一通讯作者。丁俊军教授是中山大学教育部干细胞与组织工程重点实验室独立PI,研究工作主要集中在胚胎干细胞、体细胞重编程以及早期胚胎发育的表观遗传学调控机制。丁俊军课题组长期招聘副教授、博士后,也招收博士生、硕士生等,方向为:生物信息学、干细胞、分子生物学等。References:1.         Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. 2017;18(5):285–98.2.         Sabari BR, Dall'Agnese A, Young RA. Biomolecular condensates in the nucleus. Trends Biochem Sci. 2020;45(11):961–77.3.         Kroschwald S, Maharana S, Alberti S. Hexanediol: a chemical probe to investigate the material properties of membrane-less compartments. Matters. 2017.4.         Ming Y, Chen X, Xu Y, Wu Y, Wang C, Zhang T, et al. Targeting liquid-liquid phase separation in pancreatic cancer. Transl Cancer Res. 2019;8(1):96–103.5.         Itoh Y, et al. 1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells. Life Sci Alliance. 2021;4.6.         Bonev B, Cavalli G. Organization and function of the 3D genome. Nat Rev Genet. 2016;17(11):661–78.7.         Li R, Liu Y, Hou Y, Gan J, Wu P, Li C. 3D genome and its disorganization in diseases. Cell Biol Toxicol. 2018;34(5):351–65.

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Farewell party of Qian Ma 马倩师姐送别会

On July 3rd, 2021, the whole staff of the lab held a farewell party for the graduated Doctor Qian Ma.2021年7月3日,实验室全员为毕业的马倩师姐举行了欢送会。We have prepared a cake for you, and hope your future will be plain sailing.我们为你准备了一个蛋糕,祝你未来一帆风顺。The lab members also prepared special gifts for you.实验室成员还为你准备了一些特殊礼物。Hope the time we spent together will always be in our memories.希望我们相处的时间一直留在记忆里。

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New publishment in Cell Stem Cell 在CSC新发表文章一篇

Link to this article:https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(21)00181-8The eukaryotic three-dimensional (3D) genome is organized in a hierarchical order, mainly comprising compartments, topological-associated domains (TADs), and chromatin loops from large to small scales. 3D chromatin architectures are drastically altered during cell fate transitions, which plays an important role to promote cell fate transitions. TADs are usually considered to be stable among different cell types and species. However, recent studies have reported the loss of TADs during pluripotent stem cell (PSC) differentiation, indicating that they are likely to reorganize in these biological processes. Therefore, it is significant to clarify the relationship between TAD reorganization and cell fate transitions. On 25 May 2021, the work of Professor Ding Junjun's team entitled "Phase separation of OCT4 controls TAD reorganization to promote cell fate transitions" is published in Cell Stem Cell, which for the first time illuminates that phase separation promotes cell fate transitions via regulating higher-order chromatin 3D architectures.Phase separation of OCT4 controls TAD reorganization to promote cell fate transitions In this study, 3D genome, proteome, transcriptome and epigenome were integrated to map the dynamics of chromatin 3D architectures during somatic cell reprogramming. TAD reorganization was observed, which contributes largely to cell fate transitions. Moreover, the dynamics of OCT4-mediated chromatin loops promote TAD reorganization by regulating the binding of CTCF on TAD boundaries. Further, OCT4 phase-separated condensates which concentrate chromatin loops regulate TAD reorganization. Interestingly, manipulation of TAD reorganization or OCT4 phase separation can influence cell fate transitions. Finally, TAD reorganization-based new algorithm was developed to identify novel cell fate regulators, which were validated by functional study. It is the first work to establish the regulatory network among phase separation, higher-order chromatin structures and cell fate transitions. New methods were set up to control cell fate transitions by manipulating TAD structures or phase separation. New algorithm was developed to precisely predict novel cell fate regulators. Wang Jia, research fellow of Zhongshan School of Medicine, is the first author of the paper, Yu Haopeng and Ma Qian are co-first authors, Ding Junjun, Professor of Zhongshan School of Medicine at Sun Yat-sen University, is the only corresponding author. 真核细胞染色体通常会有序的折叠,在空间上会形成有序的三维结构。这些三维结构由大到小主要分为区室分隔(compartments)、拓扑相关结构域(Topological-Associated Domains, TADs)以及染色质环状结构(loops)等【1-3】。细胞命运转变过程中往往伴随着染色体三维结构的剧烈变化,而这些变化对于推动细胞命运转变的进行起到重要作用【4-6】。TAD通常被认为是一种相对保守的结构,在不同物种和同一物种的不同细胞之间趋于稳定【2】。然而近年来的研究发现,在细胞分化的过程中,TAD的数量和大小都会发生变化【6, 8】,这说明TAD在细胞命运转变过程中有可能发生了重组。然而,这种TAD的重组是否会在细胞命运转变中发挥重要作用?是什么因素导致了TAD的重组?关键细胞命运决定因子在这一过程中又起着何种作用?这些问题都有待深入探讨和解决。2021年5月25日,来自中山大学中山医学院的丁俊军实验室在Cell Stem Cell上发表了题为 Phase separation of OCT4 controls TAD reorganization to promote cell fate transitions 的文章,首次阐明了关键转录因子OCT4通过相分离(phase separation)机制调控TAD重组以推动体细胞重编程的进行。该研究是第一篇阐述相分离机制调控染色质高级结构以推动细胞命运转变的文章。在该项研究中,研究人员首先对体细胞重编程不同时期进行了三维基因组学、蛋白组学、转录组学和表观组学等多组学的四维整合性分析,通过这些分析得到了重编程过程中以TAD重组为核心的染色体三维结构的变化特点;阐述了OCT4介导的染色体环状结构的动态变化通过改变TAD边界上CTCF的结合以调控TAD重组的新机制;进一步发现OCT4的相分离特性可调控TAD重组和细胞命运转变;进而发现,通过操控TAD重组和OCT4的相分离,可以调控细胞命运转变;最终通过基于TAD重组的新算法鉴定出了新型细胞命运调控因子,并对这些新型细胞命运调控因子做了功能验证。研究人员发现TAD重组和细胞命运转变之间存在很大的相关性。为了验证TAD重组导致细胞命运转变的因果关系,研究者通过两种方法人为诱导TAD重组,并检验对于重编程效率的影响。第一种方法是基于dCas9靶向和小分子诱导连接的方法,即通过dCas9-ABI蛋白和dCas9-PYL1蛋白分别靶向相邻TAD中的位点,由于ABI和PYL1蛋白可同时与小分子脱落酸结合,加入脱落酸分子可以形成连接(linking),这种连接可拉近TAD之间的距离,导致跨TAD边界的loop增多,最终导致了TAD的融合重组。另一种方法是通过CRISPR/Cas9的基因编辑技术敲除两个相邻TAD间的边界,使TAD发生融合重组。HiC结果表明,该两种方法均可有效造成TAD的融合重组。功能实验结果表明,人为TAD重组之后,重编程效率都发生了显著的提高,说明了TAD重组对于细胞命运转变的推动作用。研究人员还发现,OCT4介导的loop富集的区域,TAD发生重组的频率较高,说明了TAD重组和OCT4的富集存在某种相关性。考虑到OCT4富集的区域有可能会是OCT4蛋白的相分离区域,研究中通过FISH实验验证了OCT4蛋白的相分离和TAD重组的相关性。为了验证OCT4的相分离调控TAD重组的因果关系,研究人员通过两种方法破坏OCT4的相分离能力。第一种是之前报道过的酸性突变方法【9】,即将OCT4无序区(intrinsically disordered domain, IDR)中的所有酸性氨基酸突变为丙氨酸;第二种是研究人员自行开发的一种新算法PSPHunter,可以预测对相分离起到关键作用的氨基酸位点并进行删除突变。基于两种方法得到的不同的OCT4突变体均可有效的减弱OCT4蛋白的相分离能力。重要的是,这些相分离功能残缺的突变体会抑制TAD的重组和降低重编程的效率。为了进一步验证OCT4相分离调控TAD重组的因果关系,研究人员还对突变型OCT4进行了IDR融合的相分离恢复(rescue)实验。该方法是将FUS蛋白的IDR区域融合到突变型OCT4的后边,而FUS蛋白的IDR区域具有很强的相分离能力。实验验证发现,IDR融合后,突变型OCT4的相分离能力得到恢复,这种相分离能力的恢复最终导致了TAD重组的恢复和重编程效率的提高。这些结果表明,OCT4的相分离特性是推动TAD重组和细胞命运转变的关键。最后,研究人员通过基于TAD重组的多组学分析开发了预测新型细胞命运调控因子的TADMAN算法(TAD reorganization-based Multiomics Analysis, TADMAN)。通过该算法分别预测了新型重编程调控因子和新型神经分化调控因子,通过shRNA的表达敲低实验发现,超过90%的新型因子都通过了功能验证,说明了该算法的可靠性和准确性。该算法被期待在未来能应用于更为广阔的领域,如包括预测和鉴定肿瘤和衰老等在内的疾病调控因子当中。该论文首次发现蛋白质相分离、染色质三维结构和细胞命运转变之间的调控机制,建立了通过操控TAD重组、和控制蛋白质相分离,从而调控细胞命运的新方法,也开发了基于染色质三维结构精准预测细胞命运调控因子的新算法。References:1. Lieberman-Aiden, E., et al., Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science, 2009. 326(5950): p. 289-93.2. Dixon, J.R., et al., Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature, 2012. 485(7398): p. 376-80.3. Rao, S.S., et al., A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell, 2014. 159(7): p. 1665-80.4. Beagan, J.A., et al., Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming. Cell Stem Cell, 2016. 18(5): p. 611-24.5. Krijger, P.H., et al., Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming. Cell Stem Cell, 2016. 18(5): p. 597-610.6. Bonev, B., et al., Multiscale 3D Genome Rewiring during Mouse Neural Development. Cell, 2017. 171(3): p. 557-+.7. Dixon, J.R., et al., Chromatin architecture reorganization during stem cell differentiation. Nature, 2015. 518(7539): p. 331-6.8. Zhang, Y., et al., Transcriptionally active HERV-H retrotransposons demarcate topologically associating domains in human pluripotent stem cells. Nat Genet, 2019.9. Boija, A., et al., Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell, 2018. 175(7): p. 1842-1855 e16.

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Graduation of Master Student Xiaona 小娜师姐硕士毕业

PCGF6 Regulates Stem Cell Pluripotency via Super-Enhancer Dependent Chromatin Interactions        Polycomb group (PcG) ring finger protein 6 (PCGF6), known as a core component of the transcription-repressing complexes, PcG, canonically acts as a transcriptional repressor to regulate embryonic development. It also functions as a transcriptional activator to regulate the expression of pluripotency genes. However, the molecular mechanism underlying the activation function of PCGF6 remains to be further understood.       In this study, we aim to explore the molecular mechanism of PCGF6 regulating pluripotency of mouse embryonic stem cells (mESCs). We knocked down Pcgf6 and found that it was not able to maintain the phenotype of mESCs, and the Oct4-GFP positive colonies were significantly reduced in the pre-iPSC reprogramming.        Further, PCGF6 was recruited by OCT4 to multiple super-enhancer (SE) regions upstream of proliferation genes, and promoted their expression levels and then enhanced the proliferation rate of mESCs. We analyzed the expression of SE-associated genes after Pcgf6 knockdown, and found that Pcgf6 knockdown downregulated those genes. By analyzing the promoter capture Hi-C data, we showed that PCGF6 activated those proliferation genes by regulating SE-promoter interactions in 3D chromatin.       Taken together, the role of PCGF6 in regulating pluripotency of mESCs is well established. We provide a potential insight into the new molecular mechanism of PcG components.主要报告内容:PCGF6经依赖超级增强子的染色质互作调控干细胞多能性        多梳蛋白家族环指蛋白PCGF6,是转录抑制复合物的核心组分之一,它传统的功能是作为转录抑制因子来调控胚胎发育。近期也有研究表明它可以作为转录激活因子来激活多能性基因的表达。然而,对于PCGF6的激活功能的潜在分子机制仍知之甚少。       本研究中,我们探索了PCGF6调控小鼠胚胎干细胞的多能性的分子机制。我们发现敲低Pcgf6后, mESCs的多能性表型不能维持, pre-iPSC重编程中的Oct4-GFP阳性克隆也显著减少。       更进一步地,我们发现PCGF6能被OCT4招募到许多增殖相关基因上游的超级增强子区域,促进这些基因的表达,进而提高mESCs的增殖速率。Pcgf6的基因敲低也使这些超级增强子相关基因下调。通过分析Promoter capture Hi-C数据,我们还发现这些基因的表达可以通过由PCGF6在三维水平上介导形成的超级增强子-启动子相互作用而被激活。       总之,PCGF6在mESCs的多能性调控中的作用显然十分重要,为未来研究PcG组分的调控机制方面提供了潜在的新思路。

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Opening of 2019 United Group Meeting 联合组会2019开幕

The Stem Cell United Group of Sun Yat-sen University Zhongshan School of Medicine was initiated and presided by professor Junjun Ding, taking the form of a lunch and once every two weeks.中山医学院干细胞联合组会由丁俊军教授发起和主持,每两周一次,以午餐会的形式进行。干细胞与再生医学平台的实验室人员轮流在联合组会上进行课题汇报,也会邀请校外嘉宾作汇报,目的为促进各个课题组之间的交流合作。The Stem Cell United Group for fall 2019 will be scheduled as follows:2019年的干细胞联合组会日程安排如下:On May 10th, professor Peng Xiang and professor Baomin Qin, respectively reported the subject and had an in-depth discussion with the audience.5月10日,项鹏教授和秦宝明教授分别汇报了课题,与听众展开了深入讨论。

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