Tandem Tudor Domain Probes for Nanoscale Epigenetic Decoding

 描述(申请人提供)：异染色质结构和功能的调节在调节基因表达方面起着关键作用，异染色质建立和维持的缺陷与癌症、神经退行性变、发育病理和其他人类疾病有关。然而，异染色质结构的许多基本方面还知之甚少，并且与相互冲突的模型有不同的关联。这在很大程度上是由于实验的局限性。染色质生物学和分子细胞学的一个主要挑战是如何研究单个细胞中特定调控染色质结构域的大分子结构和动力学。我们已经开发了使用超分辨率定位显微镜的模型系统，通过它我们可以在纳米级分辨率下研究特定的表观遗传染色质结构。到目前为止，我们已经成功地应用该系统通过探测组合赖氨酸乙酰化的模式来可视化活性染色质结构域。在这里，我们建议将这一方法扩展到开发适合于超分辨率显微镜的遗传编码的生物探针，该探针将识别异染色质和DNA损伤修复焦点的表观遗传特征。我们的初步结果支持对两个Tudor结构域基序的关注，因此本文的研究集中在两个主要目标上：(1)我们将开发基于SetDB1的串联Tudor结构域的荧光探针的性质。基于目前的荧光图像，我们假设所看到的结构反映了异染色质的性质。我们将可视化结合的染色质结构的形态，测量它们的尺寸和密度，并比较在核周、核周室和内部核质观察到的结构。我们将测试我们的记者与已知的异染色质表观遗传标记和异染色质蛋白成分共定位的预测。我们还将测试报告结合染色质的反应与异染色质结构的实验扰动平行的预测。(2)我们将开发基于uhrf1的串联Tudor结构域的新型基因编码的荧光探针。我们将可视化结合染色质结构的形态，测量它们的尺寸和密度，并比较异染色质亚室内结构的性质。我们将通过将串联的Tudor结构域和PhD结构域结合起来进行组合识别，从而操作这些探针来识别H3K9me3和H3的游离N-末端。总之，这些目标将导致开发和表征新的探针，用于原位标记的关键表观遗传异染色质环境的超分辨率可视化。这些根本性的进展反过来有可能从根本上改进药物发现的策略，并为染色质和表观遗传调节中的病理相关缺陷产生新的治疗方式。

 DESCRIPTION (provided by applicant): The modulation of heterochromatin structure and function plays a critical role in regulating gene expression, and defects in heterochromatin establishment and maintenance are associated with cancer, neural degeneracies, developmental pathologies, and other human diseases. However, many basic aspects of heterochromatin structure are poorly understood and are variously associated with conflicting models. This is due in large measure to experimental limitations. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures and dynamics of specific regulated chromatin domains in single cells. We have developed model systems using super-resolution localization microscopy through which we can study specific epigenetic chromatin structures at nanoscale resolution. To date we have successfully applied this system to visualizing active chromatin domains by probing patterns of combinatorial lysine acetylations. Here we propose to extend this approach to develop genetically encoded bioprobes suitable for super-resolution microscopy that will recognize epigenetic features of heterochromatin and DNA damage repair foci. Our preliminary results support a focus on two tudor domain motifs and the research proposed here thus focuses on two principal aims: (1) We will exploit the properties of fluorescent probes based on the tandem tudor domain of Setdb1. Based on current epifluorescence images, we hypothesize that the structures seen reflect the properties of heterochromatin. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the structures observed at the nuclear periphery, the perinucleolar compartment, and internal nucleoplasm. We will test the prediction that our reporters colocalize with known epigenetic marks of heterochromatin and with heterochromatin protein components. We will also test the prediction that reporter-bound chromatin responds in parallel with experimental perturbation of heterochromatin structures. (2) We will develop novel genetically encoded fluorescent probes based on the tandem tudor domain of UHRF1. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the properties of structures within the heterochromatin subcompartments. We will manipulate these probes for multivalent recognition of H3K9me3 and the free N-terminal end of H3 by combining the tandem tudor domain and PHD domains for combinatorial recognition. Together, these aims will result in the development and characterization of novel probes for the super-resolution visualization of critical epigenetic heterochromatin environments labeled in situ. These fundamental advances, in turn, have the potential to radically improve strategies for drug discovery, and yield new treatment modalities for pathologies associated defects in chromatin and epigenetic regulation.