Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells
Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells
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DOI：
10.1016/j.bbagrm.2021.194725
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发表时间：
2021-06-26
影响因子：
4.7
通讯作者：
Yuan，Chongli
中科院分区：
文献类型：
--
作者：
Mendonca，Agnes;Sanchez，Oscar F.;Yuan，Chongli
基因组的3D空间组织控制基因表达和细胞功能。异染色质（HC）是染色质中致密且大部分沉默的部分，是哺乳动物细胞核中形成和维持核组织的驱动因素。它在功能上分为高度紧凑的组成型异染色质（cHC）和转录平衡的兼性异染色质（fHC）。长期以来，异染色质被认为是一种静态结构，其高度动态的性质正在慢慢地被理解和研究。HC的这些变化在细胞周期和分化过程中以不同的时间尺度发生。大多数捕获异染色质信息的方法都是静态技术，无法提供HC组织如何随时间演变的读数。由于fHC的扩散性质和缺乏特定特征，因此也难以划定特定区域（如fHC）。由于单个细胞HC组织的异质性，异染色质变化的另一种复杂性，需要进行单细胞研究。总的来说，需要活细胞相容的工具，其可以在异染色质经历重组时稳定地跟踪异染色质。在这项工作中，我们提出了一种方法来跟踪cHC和fHC的基础上与他们相关的表观遗传标志。与传统的免疫染色方法不同，我们使用小的重组蛋白探针，使我们能够通过结合到对cHC和fHC特异性的修饰，如H3K9me3，DNA甲基化和H3K27me3，来动态监测HC。我们证明了使用的探针，以遵循药物扰动诱导的单细胞水平的HC的变化。我们还使用探针集组合使用FRET为基础的方法，使我们能够跟踪独特的染色质featuresin原位同时跟踪染色质区域富集在两个选定的表观遗传修饰。
The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin featuresin situ.