Discovery of Readers and Design of Sensors To Interpret Global Histone Marks

 描述：组蛋白的翻译后修饰(PTM)在基因组生理状态的动态调节中起着至关重要的作用。调节蛋白对接到PTMS上，锚定在我们基因组上，执行特定部位的调节功能。PTM还破坏了与PTM缺失(未修饰)的组蛋白特异性结合的蛋白质的锚定。然而，人们对结合PTM较少的组蛋白的蛋白质知之甚少，尽管它们的突变会导致遗传性疾病。此外，目前关于引入/去除组蛋白PTMS的酶的知识是通过无细胞或固定细胞环境获得的，提供了关于组蛋白PTMS动态性质的较差信息。综上所述，这些都是目前对我们基因组调控缺乏了解的重要原因。这一知识鸿沟的持续存在是一个重要的问题，因为在它被填补之前，人们仍然无法理解组蛋白PTM异常导致的各种疾病的发生、发展。我们的长期目标是更好地了解PTMS在基因组调控中的作用。这种特殊的R15应用的目的是鉴定假定的能够与PTM-非组蛋白相互作用的人类蛋白质，以研究它们在各种疾病中的作用，并设计灵敏的酶传感器来报告活细胞中的酶活性。 中心假说是，在与PTM相互作用的蛋白质模块家族中，存在能够与非PTM多肽相互作用的成员，它们具有特定的序列模式。这是根据申请者实验室提供的强有力的初步数据制定的。在粗制的细胞提取物中捕捉这种相互作用，可以在无效的选择中快速筛选出敏感的传感器。我们的理论基础是，实验验证的大规模鉴定拴在PTM非组蛋白上的蛋白质将为基因组调控提供新的见解，使研究全球组蛋白标记图谱和调控复合体的定位之间的相互作用成为可能。灵敏的酶传感器的可获得性将使细胞编程机制在活的正常细胞和疾病细胞之间的作用得到比较。两个特定的目标是：1)在已知识别甲基化的赖氨酸/精氨酸的家族中鉴定未修饰的Arg/Lys读取器；2)设计对组蛋白H3-Lys4甲基酶敏感的传感器。在AIM-1中，我们测试了8种可能的人类蛋白的无PTM组蛋白结合能力，以及点突变对这种相互作用的影响，并检测了PTM对这种相互作用的影响。使用快速结合分析，我们可以推断这些相互作用。对于AIM-2，我们在备选方案库中搜索具有最高报告效率的传感器开启状态。这种方法在搜索速度上是创新的。这项研究意义重大，因为它有望从纵向上扩大对组蛋白PTMS在基因组调控中的理解。这将使疾病中组蛋白PTM异常的预防性和治疗性操作成为可能。

 DESCRIPTION: Post-translational modifications (PTMs) of histones play a crucial role in the dynamic regulation of the physiological state of our genome. Regulatory proteins dock onto PTMs for anchorage on our genome to perform site-specific regulatory functions. PTMs also disrupt the anchorage of proteins that specifically bind PTM-less (unmodified) histones. However, much less is known about proteins that bind PTM-less histones even though mutations in them cause inherited disorders. In addition, the current knowledge about the enzymes that introduce/remove histone PTMs is obtained using cell-free or fixed cell contexts providing poor information on the dynamic nature of histone PTMs. Taken together, these significantly contribute to the lack of current understanding of our genome regulation. Continued existence of this knowledge gap represents an important problem because, until it is filled, understanding the initiation, progression of various diseases due to histone PTM aberrations remain incomprehensible. Our long-term goal is to better understand the role of PTMs in genome regulation. The objective for this particular R15 application is to identify putative human proteins capable of interacting with PTM-less histones to investigate their role in various disorders, and to design sensitive enzyme sensors for reporting enzymatic activity in living cells. The central hypothesis is that in the family of protein modules that interact with PTMs, there are members capable of interacting with PTM-less peptide and they possess specific sequence patterns. This is formulated on strong preliminary data from the applicant's laboratory. Capturing such interactions in crude cellular extracts permits rapid screening of a sensitive sensor among futile alternatives. Our rationale is that experimentally verified large-scale identification of proteins that tether onto PTM-less histones will provide new insights on genome regulation, enabling studies on the interplay between global histone mark maps and the localization of regulatory complexes. The availability of sensitive enzyme sensors will enable comparison of the action of cellular programming machinery between living normal and diseased cells. The two specific aims are: 1) Identify unmodified Arg/Lys readers in the family known to recognize methylated-Lys/Arg; and 2) Design sensitive sensors for histone H3- Lys4 methylases. In Aim-1, we test PTM-less histone binding capability of 8 putative human proteins, effect of point mutations in such interactions and examine the effect of PTMs on such interaction. Using rapid binding assays we infer about these interactions. For Aim-2, we search sensor ON-state with the highest reporting efficiency among a library of alternatives. The approach is innovative for the rapidity in the searches. The research is significant, because it is expected to vertically expand the understanding of histone PTMs in genome regulation. That will enable preventative, therapeutic manipulations of histone PTM aberrations in diseases.