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 features in situ.

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) 是染色质中密集压缩和很大程度上沉默的部分，是哺乳动物细胞核中核组织形成和维持的驱动因素。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)。长期以来被视为静态结构的异染色质的高度动态性质正在慢慢被理解和研究。在细胞周期和分化过程中，HC 的这些变化发生在不同的时间尺度上。大多数捕获有关异染色质信息的方法是静态技术，无法提供 HC 组织如何随时间演变的读数。由于其扩散性质和缺乏特定特征，诸如 fHC 之类的特定区域的划分也变得困难。由于单个细胞的 HC 组织的异质性，因此需要对单细胞进行研究，从而导致表征异染色质变化的另一种复杂程度。总体，需要能够在异染色质经历重组时稳定追踪它的活细胞兼容工具。在这项工作中，我们提出了一种基于与 cHC 和 fHC 相关的表观遗传特征来跟踪它们的方法。不同于传统的在免疫染色方法中，我们使用小型重组蛋白探针，通过结合特定于 cHC 和 fHC 的修饰，例如 H3K9me3、DNA 甲基化和 H3K27me3，我们可以动态监测 HC。我们证明了使用探针来跟踪单细胞水平的药物扰动引起的 HC 的变化。我们还使用基于 FRET 的方法组合地使用探针组同时跟踪富含两种选定表观遗传修饰的染色质区域，该方法使我们能够原位跟踪独特的染色质特征。