DNA amplification in the Sciara genome

项目摘要 定点染色体内DNA扩增在许多癌症中很常见，但 启动这一过程是未知的。为了解决这个问题，我们将使用仅有的两个已知系统中的一个 这是发育中的一个正常过程，也就是在唾液腺多线染色体dna包中。 苍蝇的一部分，夏拉。我们建议阐明DNA泡芙扩增的诱导机制，利用 我们在开发一种转化和组装Sciara基因组的方法方面的最新进展。 我们提出了一个模型，其中上游增强子(DHS=DNA酶敏感位点)指定了 通过循环相互作用形成的原点。我们假设一个扩增因子(如 AS ECR=蜕皮激素受体)引起起源再复制，导致DNA扩增。为了测试这个模型，首先， 我们将通过SNS-seq和OK-seq来定位唾液腺基因组中的复制和再复制的起源。 这将识别在扩增起点唯一映射的任何共识基序(如起点的ECRE)。 4C将分析国土安全部和来源之间是否存在循环相互作用。诱变将测试顺式元件是否 (例如，DHS、起源ECRE)具有起源规范和重新复制的功能。接下来，我们将确定 通过共同作用结合顺式元件(DHS和起源)的反式作用因子(蛋白质和RNA) 免疫沉淀，通过ECR切割和运行以检测体内占位部位，并通过融合近距离标记 APEX2与ECR或ORC2(来源识别复合体)的质谱(LC-MS/MS)鉴定 与之相互作用的蛋白质。与国土安全部和/或来源相关的候选蛋白质将被敲除- 下至检测它们是否需要进行DNA扩增。这些结果将得到确认，并在 使用CRISPR将APEX2靶向国土安全部或起源地进行邻近标记的无偏方法来识别 结合在这些位点上的蛋白质。类似的定向和不偏不倚的方法将识别和测试 在国土安全部和/或来源的ncRNA。总而言之，这些实验将测试国土安全部重要的模型 原点规范，如果它这样做，通过循环交互。调节这一过程的因素(蛋白质和核糖核酸) 互动将被确定。其中许多预计会在正常复制时出现。这些实验 应该确定加入这个蛋白质复合体并触发重新复制的发育调节因子。 例如，ECR可以与复制前复合体的组件相互作用，以允许连续加载和 微晶玻璃的激活。我们的结果将为DNA泡芙扩增提供一种机制，未来的实验可以 测试这是否可以作为启动癌症DNA扩增的范例。

Project Summary Site-specific intra-chromosomal DNA amplification is common in many cancers, but the events that initiate this process are unknown. To address this question, we will use one of only two known systems where this occurs as a normal process in development, namely in the salivary gland polytene chromosome DNA puffs of the fly Sciara. We propose to elucidate the mechanism for induction of DNA puff amplification, making use of our recent advances in development of a method for Sciara transformation and assembly of the Sciara genome. We propose a model where an upstream enhancer (DHS = DNase hypersensitive site) specifies the location of an origin through a looping interaction. We posit that developmental recruitment of an amplification factor (such as EcR = ecdysone receptor) causes origin re-replication resulting in DNA amplification. To test this model, first, we will map the origins of replication and re-replication in the salivary gland genome by SNS-seq and OK-seq. This will identify any consensus motifs that map uniquely at amplification origins (such as EcREs at the origin). 4C will assay if there is a looping interaction between the DHS and the origin. Mutagenesis will test if cis-elements (e.g., DHS, origin EcREs) have functional roles for origin specification and re-replication. Next, we will identify the trans-acting factors (protein and RNA) that bind to the cis-elements (DHS and origin) by co- immunoprecipitation, CUT&RUN to assay sites of in vivo occupancy by EcR, and proximity labeling by a fusion of APEX2 with EcR or ORC2 (origin recognition complex) to identify by mass spectrometry (LC-MS/MS) the proteins that interact with them. Candidate proteins that associate with the DHS and/or origin will be knocked- down to assay if they are required for DNA amplification. These results will be confirmed and extended in an unbiased approach using CRISPR targeting of APEX2 to the DHS or origin for proximity labeling to identify proteins bound at these sites. Similar directed and unbiased approaches will identify and test the function of ncRNA at the DHS and/or origin. Together, these experiments will test the model that the DHS is important for origin specification and if it does so by a looping interaction. Factors (proteins and ncRNA) that mediate this interaction will be identified. Many of these are expected to be present at normal replication. The experiments should identify the developmentally regulated factor that joins this protein complex and triggers re-replication. For example, EcR might interact with components of the pre-replication complex to allow continual loading and activation of Mcms. Our results will suggest a mechanism for DNA puff amplification, and future experiments can test if this may serve as a paradigm for initiation of DNA amplification in cancer.