REPEAT-INDUCED HETEROCHROMATIN FORMATION IN DROSOPHILA

果蝇中重复诱导的异染色质形成

• 批准号: 9352859
• 负责人: Sarah C.R. ELGIN
• 金额: $ 30.5万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9352859
• 关键词: A-Form DNA; Ataxia; Biochemical; Biological Models; CGG repeat; Cell Line; Cells; Characteristics; Chromatin; DNA; DNA Packaging; DNA Sequence; DNA Transposable Elements; DNA Transposons; Development; Drosophila genome; Drosophila genus; Drosophila melanogaster; Drug Modelings; Elements; Embryo; Ensure; Environment; Eukaryota; Event; FMR1; Fragile X Syndrome; Friedreich Ataxia; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Genomics; Goals; Health; Hereditary Disease; Heterochromatin; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone Deacetylation; Histones; Human; Human Genetics; Impairment; Inherited; Intellectual functioning disability; Invaded; Investigation; Knowledge; Lead; Link; Mental Retardation; Methylation; Modeling; Monitor; Mothers; Movement; Mus; Mutation; Niacinamide; Patients; Pharmaceutical Preparations; Pharmacotherapy; Play; Preclinical Drug Evaluation; Proteins; RNA Interference; Reporter; Reporter Genes; Research; Retroviridae; Ribosomal RNA; Role; Site; System; Tandem Repeat Sequences; Testing; Therapeutic; Trans-Activators; Transcriptional Regulation; Trinucleotide Repeats; Triplet Multiple Birth; base; cost effective; disability; disease phenotype; epigenetic drug; epigenetic regulation; experimental study; fly; genome integrity; histone methyltransferase; inhibitor/antagonist; insight; integration site; knock-down; loss of function; mutant; novel; piRNA; screening; small molecule

The long-term goal of this research is to understand how repetitious elements, here tandem arrays in particular, are packaged into heterochromatin for silencing. The genomes of higher eukaryotes are made up primarily of repetitious DNA, including both dispersed transposable elements (TEs) and simple tandem repeats. Silencing of repetitious DNA is essential for maintaining genome integrity and regulated gene expression. However, sporadic local expansion of tandem repeats can occur, resulting in inappropriate gene silencing and consequent human genetic disability [e.g., Friedreich's ataxia (FRDA), Fragile X syndrome (FMR)]. A key objective of this proposal is to elucidate the mechanisms that lead to silencing of different types of repetitious sequences in a common genomic environment. This is being done using “landing pad” sites in the Drosophila melanogaster (fruit fly) genome, assessing the ability of different repetitious DNA sequences to trigger heterochromatin formation, as shown by silencing (variegation) of an adjacent hsp70-driven white reporter gene. We will use genetic and biochemical approaches to determine the critical trans-acting factors for silencing three different types of repeats: a single copy TE remnant (1360); a 256-copy tandem array of a foreign sequence (the 36-bp lacO element); a 310-copy tandem array of GAA, modeling the FRDA mutation, and a planned >200-copy tandem array of CGG, modeling the FMR mutation. We anticipate that HP1a will play a role in heterochromatin formation in all cases, but that other components may differ; for example, different H3K9 histone methyltransferases and/or different histone deacetylases may be utilized. To elucidate the commonalities and differences among the silencing mechanisms, we will pursue three aims. 1. Identify trans-acting factors required to silence these tandem repeats: both a targeted and an unbiased forward genetic screen will be used to identify components of the heterochromatin system required for each case of repeat-induced silencing. ChIP-qPCR experiments and experiments tethering specific components (e.g. histone modifiers) to the repeat will be used to confirm the inferences from genetics. 2. Investigate the mechanism of targeting tandem repeats for silencing. How are repeats identified? While a piRNA mechanism is likely for TE 1360, an R-loop-based mechanism has been suggested for tandem repeats, and will be investigated. 3. Determine whether tandem repeat-induced heterochromatin formation can be reversed by drug treatment. We have shown that an inhibitor of histone deacetylases (nicotinamide) reduces the silencing triggered by the lacO repeats. The fly system should prove valuable both for screening and for checking the mechanisms by which “epigenetic” drugs impact the silencing system. A deeper understanding of how tandem repeats are silenced will provide new insights into epigenetic regulation, and facilitate development of strategies to manipulate silencing for human health management.

这项研究的长期目标是了解重复的元素，这里是串联阵列 尤其是，被包装成异染色质以供沉默。高等真核生物的基因组是由 主要是重复DNA，包括分散的转座元件和简单串联 重复着。沉默重复的DNA对于维持基因组的完整性和受调控的基因至关重要 表情。然而，串联重复序列可能会发生零星的局部扩展，导致不适当的基因 沉默和由此导致的人类遗传残疾[例如，Friedreich‘s共济失调(FRDA)，脆性X综合征 (FMR)]。这项提议的一个关键目标是阐明导致沉默的机制 同一基因组环境中不同类型的重复序列。这是通过以下方式完成的 在果蝇(果蝇)基因组中的“着陆垫”位点，评估不同 重复的DNA序列，以触发异染色质的形成，如沉默(杂色) 相邻的热休克蛋白70驱动的白色报告基因。我们将使用遗传和生化方法来确定 沉默三种不同类型重复序列的关键反式作用因子：单拷贝TE残留物(1360)； 外源序列(36bp的LACO元件)的256拷贝串联阵列；GAA的310拷贝串联阵列， 模拟FRDA突变，以及计划中的&gt；200拷贝CGG串联阵列，模拟FMR突变。 我们预计HP1a在所有情况下都会在异染色质的形成中发挥作用，但其他成分 可能不同；例如，不同的H3K9组蛋白甲基转移酶和/或不同的组蛋白去乙酰基酶可能 被利用。为了阐明沉默机制之间的共性和差异，我们将继续 三个目标。1.确定沉默这些串联重复序列所需的反式作用因子：靶向和 无偏见的正向遗传筛选将用于识别异染色质系统的成分 重复诱导沉默的每一种情况都需要。芯片定量聚合酶链式反应实验与实验系留 重复序列的特定成分(例如，组蛋白修饰物)将被用来确认来自遗传学的推断。 2.研究靶向串联重复序列沉默的机制。重复序列是如何识别的？ 虽然对于TE 1360可能是piRNA机制，但是已经提出了用于串联的基于R-环的机制 重复，并将接受调查。3.确定串联重复序列是否诱导异染色质 形成可以通过药物治疗来逆转。我们已经证明了一种组蛋白脱乙酰酶的抑制剂 (烟酰胺)减少LACO重复触发的沉默。飞行系统应该被证明是有价值的 用于筛选和检查“表观遗传”药物影响沉默系统的机制。一个 更深入地了解串联重复是如何沉默的，将为表观遗传调控提供新的见解， 并促进为人类健康管理操纵沉默的战略的制定。