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Elucidation of the epigenetic mechanisms underlying transgene activation by the HSP70A promoter in Chlamydomonas reinhardtii

阐明莱茵衣藻 HSP70A 启动子转基因激活的表观遗传机制

基本信息

批准号：
212319807
负责人：
Professor Dr. Michael Schroda
金额：
--
依托单位：
Fachgebiet Molekulare Biotechnologie und Systembiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2015-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/212319807?language=en
关键词：
Elucidation epigenetic mechanisms underlying transgene

项目摘要

Transgenic approaches in higher eukaryotes often suffer from the silencing of introduced transgenes. We have found previously that the Chlamydomonas HSP70A (A) promoter counteracts transcriptional silencing of other promoters when fused upstream from these. Our current data indicate that the effect is largely mediated by heat shock elements (HSEs) and TATA-box. Moreover, as heat shock factor 1 (HSF1) at the native A promoter mediates acetylation of histones H3 and H4 to create a particularly open chromatin structure, the anti-silencing effect at downstream promoters might be mediated by HSF1. The goal of this project is to investigate in molecular detail the mechanism(s) responsible for this anti-silencing effect. For this we ask the following questions: (i) are cis-acting elements other than HSEs involved in the anti-silencing effect? If yes, which are these? (ii) Which histone modifications serving as silencing marks at transgene-driving promoters are opposed by the A promoter? (iii) Is the anti-silencing effect indeed mediated by HSF1? (iv) Is there a cascade of different histone modifications triggered via the A promoter / HSF1 that leads to the remodeled chromatin state at transgenic promoters? We propose to address these questions by combining transgenic approaches based on various A promoter mutation/deletion constructs and an inducible artificial microRNA construct targeting HSF1 with chromatin immunoprecipitation and quantitative real-time PCR. The understanding of the epigenetic mechanisms by which the A promoter counteracts transgene silencing will open up new strategies to facilitate transgenic approaches in microalgae and potentially also in other eukaryotes.

高等真核生物中的转基因方法经常遭受引入转基因沉默的困扰。我们之前发现衣藻 HSP70A (A) 启动子在与其他启动子上游融合时会抵消其他启动子的转录沉默。我们目前的数据表明，这种效应很大程度上是由热休克元件 (HSE) 和 TATA-box 介导的。此外，由于天然 A 启动子处的热休克因子 1 (HSF1) 介导组蛋白 H3 和 H4 的乙酰化以创建特别开放的染色质结构，下游启动子的抗沉默作用可能是由 HSF1 介导的。该项目的目标是从分子角度详细研究导致这种反沉默作用的机制。为此，我们提出以下问题：（i）除 HSE 之外的顺式作用元件是否参与抗沉默作用？如果是，这些是什么？ (ii) A 启动子反对哪些在转基因驱动启动子上充当沉默标记的组蛋白修饰？ (iii) 抗沉默作用确实是由 HSF1 介导的吗？ (iv) 是否存在通过 A 启动子/HSF1 触发的一系列不同组蛋白修饰，从而导致转基因启动子处染色质状态的重塑？我们建议通过将基于各种 A 启动子突变/缺失构建体和针对 HSF1 的诱导型人工 microRNA 构建体与染色质免疫沉淀和定量实时 PCR 相结合的转基因方法来解决这些问题。对 A 启动子对抗转基因沉默的表观遗传机制的理解将为促进微藻以及其他真核生物中的转基因方法开辟新的策略。