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

高等真核生物中的转基因方法经常受到引入的转基因沉默的影响。我们以前已经发现，衣藻HSP70A(A)启动子在上游融合时，可以抵消其他启动子的转录沉默。我们目前的数据表明，这种影响在很大程度上是由热休克元素(HSE)和TATA-box介导的。此外，由于天然A启动子上的热休克因子1(HSF1)介导组蛋白H3和H4的乙酰化，从而产生特别开放的染色质结构，因此下游启动子上的抗沉默作用可能是由HSF1介导的。这个项目的目标是从分子上详细研究这种抗沉默作用的机制(S)。为此，我们提出以下问题：(I)除HSE外的顺式作用元件是否参与了抗沉默效应？如果是，这些是哪几个？(Ii)A启动子反对哪些组蛋白修饰作为转基因驱动启动子的沉默标记？(Iii)抗沉默作用是否真的由HSF1介导？(Iv)是否存在通过A启动子/HSF1触发的不同组蛋白修饰的级联反应，从而导致转基因启动子的染色质状态重塑？我们建议将基于各种A启动子突变/缺失构建体的转基因方法和针对HSF1的可诱导的人工microRNA构建体与染色质免疫沉淀和实时定量聚合酶链式反应相结合来解决这些问题。对A启动子抵消转基因沉默的表观遗传学机制的理解将为在微藻和其他真核生物中促进转基因方法的发展开辟新的策略。

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.