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

 产品说明：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。调节蛋白停靠在PTM上，锚定在我们的基因组上，以执行位点特异性调节功能。PTM还破坏特异性结合无PTM（未修饰）组蛋白的蛋白质的锚定。然而，对结合PTM较少的组蛋白的蛋白质知之甚少，即使它们中的突变引起遗传性疾病。此外，目前关于引入/去除组蛋白PTM的酶的知识是使用无细胞或固定的细胞环境获得的，提供了关于组蛋白PTM的动态性质的不良信息。总的来说，这些都大大有助于我们目前缺乏对基因组调控的理解。这种知识缺口的持续存在代表了一个重要的问题，因为在它被填补之前，理解由于组蛋白PTM畸变引起的各种疾病的开始和进展仍然是不可理解的。我们的长期目标是更好地了解PTM在基因组调控中的作用。这个特定的R15应用的目的是鉴定能够与无PTM组蛋白相互作用的推定的人类蛋白质，以研究它们在各种疾病中的作用，并设计用于报告活细胞中酶活性的敏感酶传感器。 中心假设是，在与PTM相互作用的蛋白质模块家族中，存在能够与无PTM肽相互作用的成员，并且它们具有特定的序列模式。这是根据申请人实验室提供的有力的初步数据制定的。在粗细胞提取物中捕获这种相互作用允许在无用的替代品中快速筛选敏感的传感器。我们的理由是，实验验证的大规模鉴定蛋白质，系到PTM-少组蛋白将提供新的见解基因组调控，使全球组蛋白标记地图和本地化的监管复合物之间的相互作用的研究。敏感的酶传感器的可用性将使活的正常细胞和患病细胞之间的细胞编程机制的行动进行比较。这两个具体目标是：1）在已知识别甲基化-Lys/Arg的家族中鉴定未修饰的Arg/Lys读取器;和2）设计用于组蛋白H3-Lys 4甲基化酶的敏感传感器。在Aim-1中，我们测试了8种假定的人类蛋白质的无PTM组蛋白结合能力，点突变在这种相互作用中的作用，并检查PTM对这种相互作用的影响。使用快速结合试验，我们推断这些相互作用。对于Aim-2，我们搜索传感器ON状态，在替代品库中具有最高的报告效率。该方法是创新的快速搜索。这项研究意义重大，因为它有望垂直扩展对组蛋白PTM在基因组调控中的理解。这将使预防性，治疗性操纵组蛋白PTM畸变的疾病。

 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.