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

真核生物基因组的DNA通过与组蛋白和其他染色体蛋白的结合被包装成不同的形式：更浓缩的形式异染色质抑制基因表达，而更开放的形式常染色质促进基因表达。真核生物基因组在进化过程中由于重复序列的保留而大大扩展，主要是转座元件（DNA转座子和逆转录病毒- TEs）的拷贝。当这些序列高密度存在时，它们被包装成异染色质，抑制这些元素的表达。该项目的长期目标是了解基因如何从具有高水平沉默重复序列的结构域表达。在果蝇黑腹果蝇身上的研究表明，当通过重排或转位插入异染色质时，通常驻留在常染色质中的许多基因表现出多样化的表型（由于在某些应该活跃的细胞中基因沉默）。第4条小染色体（Muller F元件）大部分是异色的，包含约80个基因，既有内务管理基因，也有发育调节基因。在高等真核生物中，特别是哺乳动物，包含基因的长染色体臂的结构类似于F元素，有~30%的TE重复。因此，基因如何在这种环境中发挥作用，而重复的元素被沉默，是一个普遍的问题。这里的目标是了解使第四染色体基因在染色质环境中发挥作用的特征组合，在染色质环境中，常染色质基因多样化（沉默）。研究者假设存在一个或多个关键序列特征或基序来组织和/或保护第四染色体基因。为了验证这一假设，他们将在黑腹黑鲈的第4条染色体上确定“着陆点”位点，其中第4条染色体基因适当表达，但hsp70-white基因给出了多样化的表型，并测试这是否由基因的5'区域（转录起始位点，上游和下游都有约1kb的侧翼序列）决定。建立这一系统将使人们能够识别最小的特征，这些特征可以驱动第四个染色体异色位点上任何报告基因的充分表达。作为这项工作的补充，研究人员计划使用计算机方法在其他物种中寻找与D. melanogaster F元件相似的序列。这种比较生物信息学方法将通过开展ChIP-seq实验来寻找RNA聚合酶II的结合位点（标记转录起始位点），在两个进化距离适合寻找序列基序的果蝇物种中，D. biarmipes和D. elegans。通过用另一种方法的方法测试从一种方法获得的发现，研究人员希望更好地了解不同染色体区域的整体调控以及驱动异染色质基因表达所需的序列元件。更广泛的影响：该项目是与基因组学教育伙伴关系（GEP）的合作成果，该伙伴关系由80多名教师组成，他们正在使用果蝇基因组学将学生带入研究社区。GEP本科生将负责为D. biarmipes和D. elegans的Muller F元件上的基因生成高质量的注释。学生的注释将有助于使用比较基因组学方法搜索F元件的共同特征/基序。本科生还将负责研究Su（var）和E（var）突变对各种报告基因的影响，测试“合成第四染色体基因”结构对适当染色体蛋白质突变的敏感性。支持的博士后将成为GEP不可或缺的一员，并将非常熟练地与本科生一起开展研究项目。

Intellctual Mert: The DNA of the eukaryotic genome is packaged by association with the histones and other chromosomal proteins into alternative forms: the more condensed form, heterochromatin, inhibits gene expression while a more open form, euchromatin, facilitates gene expression. Eukaryotic genomes have been greatly expanded over evolutionary time by the retention of repetitious sequences, primarily copies of transposable elements (DNA transposons and retroviruses - TEs). Where these sequences are present at high density, they are packaged as heterochromatin, inhibiting expression of these elements. The long-term goal of this project is to understand how genes can be expressed from a domain with high levels of silenced repetitious sequence. Work in the fruit fly Drosophila melanogaster has shown that many genes normally resident in euchromatin exhibit a variegating phenotype (due to silencing of the gene in some of the cells where it should be active) when inserted into heterochromatin by rearrangement or transposition. The small fourth chromosome (Muller F element), which is largely heterochromatic, contains ~80 genes, both housekeeping and developmentally regulated genes. In higher eukaryotes, specifically mammals, the long chromosome arms that contain genes appear to be structured like the F element, with ~30% TE repeats. Thus how genes can function in this environment, while repetitious elements are silenced, is a general issue. The goal here is to understand the combination of characteristics that enables fourth chromosome genes to function in a chromatin context where euchromatic genes variegate (are silenced). The investigators postulate the presence of one or more key sequence features or motifs organizing and/or protecting the fourth chromosome genes. To test this hypothesis, they will identify 'landing pad' sites in the D. melanogaster fourth chromosome where a fourth chromosome gene is appropriately expressed, but an hsp70-white gene gives a variegating phenotype, and test whether this is determined by the 5' region of the gene (the transcription start site with ~1 kb flanking sequence, both upstream and downstream). Establishing this system will allow one to identify the minimal features that can drive full expression of any reporter in a fourth chromosome heterochromatic site. As a complement to this work, the investigators plan to use an in silico approach to find sequences in other species that are similar to the sequences identified in the D. melanogaster F element. This comparative bioinformatics approach will be facilitated by carrying out ChIP-seq experiments to find binding sites for RNA polymerase II (marking the start site for transcription) in two Drosophila species which are at a suitable evolutionary distance for finding sequence motifs, D biarmipes and D. elegans. By testing the findings gained from one approach with the methodology of the other approach, the investigators expect to gain a better understanding of the global regulation of different chromosomal regions and of the sequence elements needed to drive expression of genes in heterochromatin.Broader Impacts: This project is a collaborative effort with the Genomics Education Partnership (GEP), a group of over 80 faculty who are using Drosophila genomics to bring students into the research community. The GEP undergraduate students will be responsible for generating high quality annotations for the genes on the Muller F elements of D. biarmipes and D. elegans. The student annotations will facilitate the search for common features/motifs on the F element using a comparative genomics approach. Undergraduates will also take charge of looking at the impact of Su(var) and E(var) mutations on various reporters, testing sensitivity of the "synthetic fourth chromosome gene" constructs to mutations in appropriate chromosomal proteins. The postdoctoral fellow supported here will become an integral member of GEP, and will become very skilled at working with undergraduates in a research program.