DNA Amplification in the Sciara Genome

位点特异性染色体内DNA扩增在许多癌症中是常见的，但是， 启动该过程的事件是未知的。为了解决这个问题，我们将使用其中一个 两个已知的系统，其中这作为开发中的正常过程发生，即在 唾液腺多线染色体DNA粉扑的苍蝇Sciara。我们建议澄清 诱导DNA喷烟扩增的机制，利用我们最近的进展， - 开发用于Sciara转化、Sciara基因组组装的方法，以及 在基因组序列中识别DNA泡沫。首先我们将绘制复制的起源 和唾液腺基因组中的再复制，使用NS-seq和ORC-ChIP以及 通过DNA梳理验证再复制起点。接下来，我们将识别共识基序 在扩增起点附近作图，并通过缺失诱变以及 通过放置在通常不会重新复制的原点进行功能测试，以询问它现在是否 被迫重新复制有了顺式调控元件，我们将确定反式作用元件。 因子（蛋白质和RNA）结合这些元素的下拉实验与体内 ChIP的占用验证。为了进行功能测试，我们将诱导性地连接 因子到通常不重新复制的起源，以询问它现在是否被驱动重新复制。 这将包括开发用于诱导系留RNA的方法，如果反式作用的话。 因子是ncRNA。在本资助申请的最后一部分，我们将使用反作用因子 用于下拉实验，以确定与之相互作用的蛋白质。在许多可能性中， 这种相互作用蛋白质可以是复制机制的组成部分， 持续加载和激活Mcms或修改染色质环境。我们的结果将 提出了一种DNA蓬松扩增的机制，未来的实验可以测试这是否可以 作为癌症中DNA扩增起始的范例。

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, assembly of the Sciara genome, and identification of DNA puffs in the genome sequence. First we will map the origins of replication and re-replication in the salivary gland genome, using NS-seq and ORC-ChIP as well as validation of the re-replication origins by DNA combing. Next, we will identify consensus motifs that map near the amplification origins and test them by deletion mutagenesis as well as a functional test by placement at an origin that normally does not re-replicate to ask if it is now driven to re-replicate. With the cis-regulatory elements in hand, we will identify the trans-acting factors (protein and RNA) that bind to these elements by pull-down experiments with in vivo occupancy validation by ChIP. For a functional test, we will inducibly tether the trans-acting factor to an origin that normally does not re-replicate to ask if it is now driven to re-replicate. This will include development of methodology for induction of tethered RNA, if the trans-acting factor is a ncRNA. In the final section of this grant application, we will use the trans-acting factor for pull-down experiments to identify proteins that interact with it. Among many possibilities, such interacting proteins may be components of the replication machinery or modify it to be continually load and activate Mcms or modify the chromatin environment. 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.