The Drosophila Dot Chromosome: Gene Expression in the Context of Repetitious DNA

真核生物的染色体包含紧密排列的区域，称为异染色质。这些区域的基因通常是沉默的；然而，异染色质区域的一些基因是活跃表达的。这个项目的目的是解决基因调控领域中这个长期存在的重要问题：是什么机制允许位于异染色质区域的某些基因表达？这项工作是与基因组教育伙伴关系的学生和教职员工共同努力的结果。来自60多所高校的本科生将分析几种果蝇第四染色体上的异染色质DNA序列。这项工作的目标是确定异染色质DNA中基因表达的潜在调控基序。这个过程将允许学生发展基本的生物信息学技能，参与基因组学研究，并成为最终出版物的撰稿人(和合著者)。果蝇的第四条染色体具有30%的重复密度，在很大程度上是异染色质；正如第四条染色体上大多数插入点的HSP70-白色报告基因的多样性表型所表明的那样。这种沉默似乎是由于异色形式的包装造成的。虽然在第四染色体基因体内发现高水平的HP1a和H3K9me2/3，但它们的转录起始点(TSS)始终显示这些沉默标记的耗尽。该项目在第四条染色体上发现了一个“着陆垫”(一个模拟线)，在那里插入的HSP70-白色转基因产生了多样化的表型，而用荧光标记标记的第四条染色体基因(RAD23)显示了充分的表达。这些构建体之间的基因片段将被交换，以确定第四条染色体基因的哪些特征在这种异染色质环境中充分表达，最初的重点是TSS周围的区域。该项目的目标是确定在这个染色质位置驱动HSP70-白色充分表达所需和足够的序列元件，以及相反地，那些会导致RAD23多样性的变化(参与转录延伸的元件可能被证明与TSS一样重要，不会被忽视)。生物信息学方法确定的关键主题将在该系统中进行测试，反之亦然。不同品系的染色质结构将通过遗传和生化方法(分析SU(Var)突变的影响；芯片聚合酶链式反应实验)进行检查。这些研究将有助于更好地理解异染色质基因的调控，并识别影响富重复区域基因表达的基序(其中一些可能是新的)，这些重复结构域在高等真核生物基因组中也很常见。

The chromosomes of eukaryotes contain tightly packed regions known as heterochromatin. Genes in these regions are typically silenced; however, some genes in heterochromatic regions are actively expressed. The aim of this project is to address this long-standing and important question in the field of gene regulation: what is the mechanism that allows certain genes located in heterochromatic regions to be expressed? This work is a collaborative effort with the students and faculty of the Genomics Education Partnership. Undergraduate students from over 60 colleges and universities will analyze heterochromatic DNA sequences in the fourth chromosome of several fruit fly species. The goal of this work is to identify potential regulatory motifs for gene expression in heterochromatic DNA. This process will allow the students to develop basic bioinformatics skills, participate in genomics research, and be contributors (and co-authors) on the resulting publications. The fourth chromosome of Drosophila melanogaster, with a 30% repeat density, is largely heterochromatic; as illustrated by the variegating phenotype of an hsp70-white reporter gene at most insertion sites on the fourth chromosome. This silencing appears to be due to packaging in a heterochromatic form. While high levels of HP1a and H3K9me2/3 are found across the body of fourth chromosome genes, their transcription start sites (TSSs) consistently show depletion of these silencing marks. This project hase identified a "landing pad" site (a MiMIC line) on the fourth chromosome where an inserted hsp70-white transgene gives a variegated phenotype, while a fourth chromosome gene (Rad23) tagged with a fluorescent marker shows full expression. Gene fragments between these constructs will be swapped to determine what features of the fourth chromosome gene drive full expression in this heterochromatic environment, with an initial focus on the region around the TSS. The goal of this project is to determine the sequence elements necessary and sufficient to drive full expression of hsp70-white at this chromatin site, and conversely those changes that would result in variegation of Rad23 (Elements involved in transcription elongation may prove to be as important as the TSS, and will not be neglected). Key motifs identified by the bioinformatics approach will be tested in this system, and vice-versa. The chromatin structure of the various lines will be checked by both genetic and biochemical approaches (analysis of the impact of Su(var) mutations; ChIP-PCR experiments). These studies will lead to a better understanding of heterochromatic gene regulation, and identification of motifs (some of which may be novel) that influence gene expression in repeat-rich domains that are also common in the genomes of higher eukaryotes.