Investigation of the mechanisms by which the HSP70A promoter counteracts transcriptional gene silencing in Chlamydomonas

高等真核生物中的转基因方法经常遭受引入的转基因的沉默。我们最近发现，衣原体HSP70A启动子可以抵消其他启动子的转录沉默时，这些融合的上游。这种作用是由两个独立的序列元件内的HSP70A启动子，最有可能，通过吸引染色质重塑活动。该项目的目标是在分子上详细研究这一发现的潜在机制。为此，我们将首先确定HSP70A启动子可能对染色质结构的影响。接下来，我们要确定两个HSP70A启动子元件内的核心基序，并纯化结合（激活）蛋白。此外，我们将分析与这些激活剂相互作用的蛋白质复合物。最后，我们希望阐明哪些衣原体基因需要这些激活剂才能正常表达。从长远来看，所获得的数据将用于建立合成启动子的设计概念，这些合成启动子不易于沉默，从而保证衣原体和高等生物中的正常转基因表达。 评审小组的意见（包括一份书面评论）：Schroda博士是一位年轻的研究人员，一年多前成立了自己的小组。Schroda博士提出的项目建立在HSP70A启动子能够克服其他启动子沉默的观察基础上。这部作品已经由他出版。在HSP70A启动子内，可以鉴定出对于拮抗作用重要的两个结构域。这里建议的项目试图更详细地调查这些有趣的发现。工作假设是HSP70A启动子结合染色质重塑成分。列出了七种不同的方法，应该解决这个假设。审查小组对调查员的热情、为建议的项目提供的坚实基础、他自己的准备工作以及对不同方法的全面概述印象深刻。审稿人唯一担心的是，Schroda博士可能会从一开始就指导两名博士生和所有拟议的方法。因此，建议最初只授予一个博士学位，消耗品预算应减少到20，000。EUR.

Transgenic approaches in higher eukaryotes often suffer from the silencing of the introduced transgenes. We have found recently that the Chlamydomonas HSP70A promoter may counteract transcriptional silencing of other promoters when fused upstream of these. This effect was mediated by two independent sequence elements within the HSP70A promoter that, most likely, act by attracting chromatin remodeling activities. The goal of this project is to investigate in molecular detail the underlying mechanism(s) of this finding. For this, we will first determine the effects that the HSP70A promoter may have on chromatin structure. Next, we want to identify the core motifs within the two HSP70A promoter elements and to purify the binding (activator) proteins. Furthermore, we will analyse the protein complexes interacting with these activators. At last, we want to elucidate which of the Chlamydomonas genes require these activators for their proper expression. On a long term, the data obtained will be used to establish concepts for the design of synthetic promoters which are less prone to silencing and, thus, guarantee normalized transgene expression in Chlamydomonas and higher organisms. Opinion of the review panel (including one written comment): Dr. Schroda is a young researcher who started his own group little more than a year ago. The project proposed by Dr. Schroda builds upon the observation that the HSP70A promoter is able to overcome a silencing of other promoters. This work has already been published by him. Within the HSP70A promoter two domains could be identified which are important for the antagonistic effect. The project suggested here attempts to investigate these interesting findings in more detail. The working hypothesis is that the HSP70A promoter binds chromatin-remodelling components. Seven different approaches are listed that should address this hypothesis. The review panel was impressed by the enthusiasm of the investigator, by the solid basis provided for the suggested project, by his own prework and by the thorough outline of the different approaches. The only concern of the reviewers was that Dr. Schroda might be overwhelmed by directing two PhD students and all the proposed approaches right from the beginning. It was, therefore, recommended that initially only one PhD position should be granted and that the budget for consumables should be reduced to 20,000.- EUR.