端粒上的化学分化诱导蛋白凝结物
Summary
该协议说明了化学诱导的蛋白质变暗系统，在染色质上产生凝结物。 用化学调光剂在端粒上形成前列腺白血病（PML）核体。滴生长、溶解、定位和组成通过活细胞成像、免疫荧光 （IF） 和荧光就地杂交 （FISH） 进行监测。
Abstract
与铬素相关的冷凝水与许多核过程有关，但其基本机制仍然难以捉摸。该协议描述了一个化学诱导的蛋白质变暗系统，在端粒上产生凝结物。化学分光器由两个连结的配体组成，每个配体可以与蛋白质结合：光环配体与光环酶和三叶虫（TMP）到 大肠杆 菌二氢脱氢还原酶（eDHFR）。光环酶与端粒蛋白的融合通过共价光环配体酶结合将调光剂锚定到端粒。TMP 与 eDHFR 结合，将 eDHFR 融合相分离蛋白到端粒，并诱导凝结物形成。由于 TMP-eDHFR 相互作用是非共价的，因此可以通过使用多余的免费 TMP 与变音器竞争 eDHFR 绑定来逆转冷凝。显示了在端粒上诱导产卵性白血病 （PML） 核体形成并确定凝结水生长、溶解、定位和成分的示例。这种方法可以很容易地适应诱导冷凝水在其他基因组位置通过融合光环到蛋白质，直接结合到当地的色素或dCas9，这是针对基因组位置与引导RNA。通过提供单细胞活成像所需的时间分辨率，同时保持生化测定细胞群的相分离，该方法适用于探索染色素相关凝结物的形成和功能。
Introduction
许多蛋白质和核酸经过液态相分离（LLPS），并自行组装成生物分子凝结物，在细胞1、2中组织生物化学。铬素结合蛋白的LLPS导致凝结物的形成，与特定的基因组位点相关，并涉及各种局部染色质功能3。例如，HP1蛋白的LLPS是异色素域形成的基础，组织基因组4、5，转录因子的LLPS形成转录中心来调节转录6，新生的mRNA和多性别梳蛋白的LLPS产生希石细胞体来调节histonemRNAS7的转录和处理。 然而，尽管发现了许多与染色素相关的冷凝水的例子，但冷凝水形成、调节和功能的基本机制仍然鲜为人知。特别是，并非所有的染色质相关冷凝水都是通过LLPS形成的，还需要仔细评估活细胞中的凝结物形成。例如，小鼠体内的HP1蛋白在活细胞中形成液滴的能力较弱，异色素foci表现为坍塌的聚合物球10。因此，在活细胞中诱导染色素的脱凝析剂的工具是可取的，特别是那些允许使用活成像和生化检测来监测凝结物形成的动能、产生的凝结水的物理和化学特性以及凝结水形成的细胞后果的工具。
该协议报告了一种化学变质系统，在染色质11（图1A）上诱导蛋白质凝结物。分光器由两个链接的蛋白质相互作用的配体组成：三叶草（TMP）和光环配体，可以调低融合到认知受体的蛋白质：大肠杆菌二氢磷酸还原酶（eDHFR）和细菌烷基卤酶酶（哈洛酶），分别12。光环配体和光环酶之间的相互作用是共价的，允许光环酶通过将其与染色质结合蛋白融合来吸收融合到eDHFR到染色质的相分离蛋白来用作锚。在初始招募后，相分离蛋白的局部浓度增加，超过了相分离所需的临界浓度，从而在锚点核聚水（图1B）。通过将荧光蛋白（例如 mCherry 和 eGFP）与 eDHFR 和光环融合在一起，冷凝水的核和生长可以通过荧光显微镜实时可视化。由于 eDHFR 和 TMP 之间的相互作用是非共价的，因此可以添加多余的免费 TMP，以与变色器竞争 eDHFR 绑定。然后，这将释放锚的相分离蛋白，并溶解与染色素相关的冷凝水。
我们用这个工具诱导端粒酶阴性癌细胞端粒上的非核细胞白血病（PML）核体形成，使用端粒（ALT）通路的替代加长端粒维持13，14。 PML核体是涉及许多核过程的无膜隔间15，16，并具有独特的本地化ALT端粒形成APB，为ALT端粒相关的PML体17，18。端粒聚集在APB内，大概是为了提供修复模板的同源定向端粒DNA合成在ALT19。事实上，端粒DNA合成已经在APB中被发现，APB在丰富端粒20、21的DNA修复因子方面起着至关重要的作用。然而，APB 组装和端粒聚类在 APB 内的基础机制尚不得而知。由于ALT细胞中的端粒蛋白由小的全非基蛋白样修饰剂（SUMOs）22独特地修饰，许多APB组件包含相扑位点22、23、24、25和/或 相扑相互作用主题（SIM）26，27和 SUMO-SIM 相互作用驱动相分离28，我们假设端粒上的相扑导致富集 SUMO/SIM 包含蛋白质和这些蛋白质之间的 SUMO-SIM 相互作用导致相位分离。 PML蛋白，它有三个相扑位点和一个SIM网站，可以招募到相扑端粒形成APB和液体APB的聚合导致端粒聚类。为了验证这一假设，我们使用化学调光系统模仿相扑诱发的APB形成，通过招募SIM到端粒（图2A）11。GFP 融合到光环酶中用于可视化，并融合到端粒结合蛋白 TRF1 上，将调光器固定到端粒上。SIM 融合到 eDHFR 和 mcherry。凝结物形成和液滴融合诱导端粒聚类的动力学与活细胞成像相跟随。 阶段分离通过添加多余的免费 TMP 来与 eDHFR 绑定竞争来逆转。免疫荧光 （IF） 和荧光原位杂交 （FISH） 用于确定凝结水成分和端粒关联。招聘 SIM 丰富端粒上的 SUMO，诱导的冷凝水含有 PML，因此是 ABB。招募不能与 SUMO 交互的 SIM 突变体不会丰富端粒上的 SUMO 或诱导相分离，表明 APB 凝结的根本驱动力是 SUMO-SIM 交互。同意这一观察，聚SUMO-聚氨酯聚合物融合到TRF2结合因子RAP1也可以诱导APB形成29。与聚SUMO-聚氨酯聚变系统相比，只要产生足够的蛋白质，相分离就会发生，这里提出的化学调光方法可按需诱导相分离，从而提供更好的时间分辨率来监测相分离和端粒聚类过程的动能。此外，这种化学分化系统允许招募其他蛋白质来评估它们诱导相分离和端粒聚类的能力。例如，在端粒中招募的无序蛋白质也可以形成液滴和聚类端粒，而不会诱导APB的形成，这表明端粒聚类独立于APB化学，仅依赖于APB液体特性11。
Protocol
Representative Results
Discussion
该协议演示了端粒上凝结水的形成和溶解与化学变暗系统。通过实时成像监测相位分离和液滴融合诱导端粒聚类的动力学。凝结物的本地化和成分由DNA鱼和蛋白质IF确定。
此协议中有两个关键步骤。首先是确定蛋白质和调光剂的浓度。在基因组位点诱导局部相分离的成功取决于相分离蛋白的局部浓度高于相分离的关键浓度（图 1B).分离蛋白的全球浓度需?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家卫生研究院（1K22CA23763201至H.Z.，GM118510至D.M.C）和查尔斯·考夫曼基金会对H.Z.的支持。作者要感谢杰森·托恩斯对手稿的校对。
Materials
| 0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate | Corning | MT25053CI | |
| 16% Formaldehyde (w/v), Methanol-free | Thermo Scientific | 28906 | Prepare 1% in 1x PBS |
| 6 Well Culture Plate | VWR | 10861-554 | |
| Aluminum Foil | Fisher Scientific | 01-213-101 | |
| Anti-mCherry antibody | Abcam | Ab183628 | |
| Anti-PML antibody | Santa Cruz | sc966 | |
| Anti-SUMO1 antibody | Abcam | Ab32058 | |
| Anti-SUMO2/3 antibody | Cytoskeleton | Asm23 | |
| Blocking Reagent | Roche | 11096176001 | |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP9706100 | |
| BTX Tube micro 1.5ML | VWR | 89511-258 | |
| Circle Cover Slips | Thermo Scientific | 3350 | |
| Confocal microscope | Nikon | MQS31000 | |
| DAPI | Fisher Scientific | D1306 | |
| Dimethyl Sulphoxide | Sigma-Aldrich | 472301 | |
| DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate | Corning | MT10017CV | |
| EMCCD Camera | iXon Life | 897 | |
| Ethanol | Fisher Scientific | 4355221 | |
| Fetal Bovine Serum, Qualified, USDA-approved Regions | Gibco | A4766801 | |
| Formamide, Deionized | MilliporeSigma | 46-101-00ML | |
| Goat anti-Mouse IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647 | Invitrogen | A28181 | |
| Goat anti-Rabbit IgG (H+L), Recombinant Secondary Antibody, Alexa Fluor 647 | Invitrogen | A32733 | |
| High Precision Straight Tapered Ultra Fine Point Tweezers/Forceps | Fisher Scientific | 12-000-122 | |
| Laser merge module | Nikon | NIIMHF47180 | |
| Leibovitz's L-15 Medium | Gibco | 21083027 | |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668027 | |
| Figure plotting software, MATLAB | The MathWorks | ||
| Microscope Slide Box | Fisher Scientific | 34487 | |
| Nail Polish | Fisher Scientific | 50-949-071 | |
| Imaging software, NIS-Elements | Nikon | ||
| Opti-MEM Reduced Serum Media | Gibco | 51985091 | |
| Parafilm | Bemis | 13-374-12 | |
| PBS 10x, pH 7.4 | Fisher Scientific | 70-011-044 | |
| Penicillin-Streptomycin Solution,100X | Gibco | 15140122 | |
| Piezo Z-Drive | Physik Instrumente (PI) | 91985 | |
| Pipet Tips | VWR | 10017 | |
| Plain and Frosted Clipped Corner Microscope Slides | Fisher Scientific | 22-037-246 | |
| Poly-D-Lysine solution | Sigma-Aldrich | A-003-E | |
| Sodium Azide | Fisher Scientific | BP922I-500 | |
| Spinning disk | Yokogawa | CSU-X1 | |
| Square Cover Slips | Thermo Scientific | 3305 | |
| TBS 10x solution | Fisher Scientific | BP2471500 | |
| TelC-Alexa488 | PNA Bio | F1004 | |
| TMP | Synthesized by Chenoweth lab | Available upon request | |
| TNH | Synthesized by Chenoweth lab | Available upon request | |
| Tris Solution | Fisher Scientific | 92-901-00ML | |
| Triton X-100 10% Solution | MilliporeSigma | 64-846-350ML | Prepare 0.5% in 1x PBS |
| U2Os cell line | From E.V. Makayev lab (Nanyang Technological University, Singapore) | HTB-96 | |
| VECTASHIELD Antifade Mounting Medium | Vector Laboratories | NC9524612 |
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