通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像
1Belozersky Institute of Physico-chemical Biology,Lomonosov Moscow State University, 2Faculty of Biology,Lomonosov Moscow State University, 3Faculty of Bioengineering and Bioinformatics,Lomonosov Moscow State University, 4National Research Center, Kurchatov Institute, 5V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology, and Perinatology
Summary
该协议提出了一种在结构上保存的染色质 中原位 复制位点的高分辨率映射技术，该技术采用预包埋EdU-链霉亲和素 – 纳米金标记和ChromEMT的组合。
Abstract
细胞核中DNA折叠的原理及其在基本遗传功能（转录，复制，分离等）实现过程中发生的动态转化仍然知之甚少，部分原因是缺乏实验方法来高分辨率地可视化结构上保存的细胞核中的特定染色质位点。在这里，我们提出了一种方案，用于 原位单层细胞培养中复制结构域的可视化，通过将新合成DNA的EdU标记与随后的标记检测与纳米金颗粒的Ag扩增和染色质的ChromEM染色相结合。该协议允许高对比度，高效率的预包埋标记，与传统的戊二醛固定兼容，为室温样品处理提供染色质的最佳结构保存。预嵌入标记的另一个优点是可以预先选择感兴趣的单元格进行切片。这对于分析异质细胞群，以及与电子断层扫描方法的兼容性，以高分辨率3D分析复制位点的染色质组织，以及分析复制后染色质重排和相间姐妹染色单体分离尤为重要。
Introduction
DNA复制是在细胞分裂过程中忠实复制和传递遗传信息所需的基本生物学过程。在高等真核生物中，DNA复制受到严格的时空调节，这表现为复制起源1的顺序激活。同步触发的相邻复制源形成 replicon2 的簇。在光学显微镜水平上，正在进行的DNA复制的位点被检测为各种数量和大小的复制焦点。复制病灶显示细胞核内空间分布的特定模式，这取决于标记的DNA3，4的复制时间，这反过来又与其基因活性密切相关。由于明确定义的DNA复制序列，在空间和时间上严格排序，复制标记是一种强大的精确DNA标记方法，不仅用于研究复制过程本身，还用于区分具有定义的转录活性和压实水平的特定DNA亚级分。复制染色质的可视化通常通过检测DNA复制机制的主要蛋白质成分（通过免疫染色或通过荧光蛋白标签5，6的表达）或通过掺入修饰的DNA合成前体7，8，9，10进行.其中，只有基于将修饰的核苷酸掺入新复制的DNA中的方法才能在复制过程中捕获染色质的构象变化，并在复制完成后跟踪复制结构域的行为。
在高等真核生物中，DNA包装成染色质为基本遗传功能（转录，复制，修复等）的调节增加了另一个层次的复杂性。染色质折叠影响DNA对调节反式因子的可及性和模板合成所需的DNA构象变化（双螺旋放卷）。因此，人们普遍认为，细胞核中DNA依赖性的合成过程需要染色质的结构转变，从其浓缩的抑制状态到更容易获得的开放构象。在细胞学上，这两种染色质状态被定义为异染色质和真染色质。然而，关于细胞核中DNA折叠的模式仍然没有达成共识。假设的范围从“聚合物熔体”模型11，其中核小体纤维表现为随机聚合物，其堆积密度由相分离机制控制，到分层折叠模型假设连续形成厚度增加12，13的染色质纤维样结构。分层折叠模型最近获得了基于 原位 DNA-DNA接触分析的分子方法的支持（染色体构象捕获，3C），证明了染色质结构域层次结构的存在14。重要的是要注意，复制单元与这些染色质结构域15的相关性非常好。对这些模型的主要批评是基于样品制备程序引起的潜在的人工染色质聚集，例如细胞膜的透化和非染色质成分的去除，以改善超微结构研究的染色质对比度，同时提高染色质对各种探针（例如抗体）的可及性。最近通过DNA结合荧光团介导的二氨基联苯胺光氧化（ChromEMT6）进行选择性DNA染色电子显微镜检查的技术进展已经消除了这一障碍。然而，同样的考虑也适用于复制DNA17，18的电子显微镜可视化。在这里，我们描述了一种技术，该技术允许在完整的醛交联细胞中同时对新合成的DNA和总染色质进行高分辨率超微结构图谱。该技术通过点击化学检测EdU标记的DNA与生物素化探针和链霉亲和素纳米金以及ChromEMT相结合。
Protocol
该方案针对贴壁细胞进行了优化，并在HeLa，HT1080和CHO细胞系上进行了测试。 1. 细胞标记和固定 在3厘米培养皿中酸性清洁的盖玻片上板细胞。将推荐用于细胞系的培养基中的细胞生长至70%汇合度。 从10mM储备液中加入EdU（5-乙炔基-2′-脱氧尿苷）至10μM终浓度，并将细胞置于培养箱中10分钟或更长时间（取决于实验目的）。对于较短的脉冲（长达2分钟…
Representative Results
哺乳动物细胞核中的复制病灶根据S期进展在细胞核内显示出不同的分布模式。这些模式与被复制的位点的转录活性相关。由于这里介绍的方法使用相当强的固定程序，因此使用复制脉冲标记来特异性检测各种转录状态下的染色质位点是相当简单的，即使在提供通过室温化学固定获得的染色质的最佳结构保存的条件下也是如此。 质量控制步骤旨在确保成功完成该协议中的关键?…
Discussion
与以前发布的协议相比，此处描述的方法具有几个优点。首先，使用点击化学标记复制的DNA消除了用抗体检测BrdU的DNA变性先决条件的必要性，从而更好地保留了染色质超微结构。
其次，利用生物素作为戊二醛固定和未结合醛基团适当淬火后产生的二级配体，最大限度地减少了靶标的化学修饰，从而提高了标记效率，同时减少了内源性生物素引起的背景。生物素 – 链霉亲和素…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了RSF（拨款#17-15-01290）和RFBR（拨款#19-015-00273）的部分支持。作者感谢莫斯科罗蒙诺索夫国立大学发展计划（PNR 5.13）和别洛泽尔斯基物理化学生物学研究所的尼康相关成像卓越中心对成像仪器的使用。
Materials
| Reagent | |||
| 5-ethynyl-2`-deoxyuridine (EdU) | Thermo Fisher | A10044 | |
| 2-(4-Morpholino)ethane Sulfonic Acid (MES) | Fisher Scientific | BP300-100 | |
| AlexaFluor 555-azide | Termo Fisher | A20012 | |
| biotin-azide | Lumiprobe | C3730 | |
| Bovine Serum Albumine | Boval | LY-0080 | |
| DDSA | SPI-CHEM | 26544-38-7 | |
| DMP-30 | SPI-CHEM | 90-72-2 | |
| DRAQ5 | Thermo Scientific | 62251 | |
| Epoxy resin monomer | SPI-CHEM | 90529-77-4 | |
| Glutaraldehyde (25%, EM Grade) | TED PELLA, INC | 18426 | |
| Gum arabic | ACROS Organics | 258850010 | |
| Magnesium chloride | Panreac | 141396.1209 | |
| NaBH4 | SIGMA-ALDRICH | 213462 | |
| NMA | SPI-CHEM | 25134-21-8 | |
| N-propyl gallate | SIGMA-ALDRICH | P3130 | |
| PBS | MP Biomedicals | 2810305 | |
| Silver lactate | ALDRICH | 359750-5G | |
| Streptavidin-AlexaFluor 488 conjugate | Termo Fisher | S11223 | |
| Streptavidin-Nanogold conjugate | Nanoprobes | 2016 | |
| tetrachloroauric acid | SIGMA-ALDRICH | HT1004 | |
| Tris(hydroxymethyl)aminomethane (Tris) | CHEM-IMPEX INT'L | 298 | |
| Triton X-100 | Fluka Chemica | 93420 | |
| Instruments | |||
| Carbon Coater | Hitachi | ||
| Copper single slot grids | Ted Pella | 1GC10H | |
| Cy5 fluorescence filter set (Ex620/60 DM660 Em700/75) | Nikon | Cy5 HQ | Alternatives: Zeiss, Leica, Olympus |
| Diamond knife Ultra Wet 45o | Diatome | DU | Alternatives: Ted Pella |
| Fluorescent microscope | Nikon | Ti-E | Alternatives: Zeiss, Leica, Olympus |
| High-tilt sample holder | Jeol | ||
| Rotator | Biosan | Multi Bio RS-24 | |
| Transmission electron microscope operating at 200 kV in EFTEM mode, with high-tilt goniometer | Jeol | JEM-2100 | Alternatives: FEI, Hitachi |
| Tweezers | Ted Pella | 523 | |
| Ultramicrotome | Leica | UltraCut-E | Alternatives: RMC |
| Software | |||
| Image acquisition | Open Source | SerialEM (https://bio3d.colorado.edu/SerialEM/) | |
| Image processing | Open Source | IMOD (https://bio3d.colorado.edu/imod/) |
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Citar este artigo
Sosnovskaya, S., Zakirov, A. N., Ryumina, E. D., Kharybina, E., Golyshev, S. A., Strelkova, O. S., Zhironkina, O. A., Moiseenko, A., Orekhov, A., Kireev, I. I. Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography. J. Vis. Exp. (183), e62803, doi:10.3791/62803 (2022).