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)00181-8


The 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.

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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。


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