Targeting of Heterochromatin Assembly in Schizosaccharomyces pombe

描述(申请人提供)：异染色质参与调节广泛的细胞过程，包括基因表达、染色体分离和细胞分化。然而，将异染色质靶向基因组特定区域的机制还不完全清楚。当我们发现从异染色质着丝粒序列合成的双链RNA被RNAi机制快速处理时，我们对这个问题有了深入的了解，从而导致了异染色质对着丝粒的序列特异性靶向。这项提议的主要目标是确定基因组的特定区域如何成为异染色质组装的靶标。我们的具体目标是：1)鉴定对异染色质组装重要的着丝粒重复序列。虽然已知着丝粒重复序列足以招募异染色质，但确切地说，这些着丝粒序列中的哪些对异染色质组装是重要的，以及它们如何发挥功能来招募沉默尚不清楚。我们将通过分析各种着丝粒重复序列将异染色质组装到异位等染色质部位的能力来直接测试这一点。2)确定RNA介导的异染色质形成是在顺式还是反式中正常发生。我们将讨论位于分裂酵母异染色质区域的着丝粒重复序列是否具有招募顺式异染色质组装的功能，或者更确切地说，是通过沉默信号作为异染色质靶向的靶标，例如在反式中产生的着丝粒小RNA。3)确定启动异染色质组装的相关因素。在具体目标3中，我们将使用一系列策略来确定启动异染色质组装所涉及的新因素。异染色质被限制在离散的染色体区域是很重要的，因为不适当的靶向异染色质可能导致基因表达的错误调节和人类疾病的发生，如癌症。我们的研究可能导致允许操纵异染色域的技术的发展，这不仅将非常有用地作为研究基因功能的实验工具，而且还将作为潜在的人类疾病治疗方法。

DESCRIPTION (provided by applicant): Heterochromatin has been implicated in the regulation of a wide range of cellular processes including gene expression, chromosome segregation and cellular differentiation. The mechanisms involved in targeting heterochromatin to specific regions of the genome, however, are not fully understood. We gained insight into this problem when we found that double stranded RNA synthesized from heterochromatic centromeric sequences becomes rapidly processed by the RNAi machinery resulting in sequence specific targeting of heterochromatin to centromeres. The main goal of this proposal is to determine how specific regions of the genome become targeted for heterochromatin assembly. Our specific aims are to: 1) Identify the centromeric repeat sequences that are important for heterochromatin assembly. Although it is known that centromeric repeats are sufficient to recruit heterochromatin, precisely which of these centromeric sequences are important for heterochromatin assembly and how they function to recruit silencing is not clear. We will directly test this by assaying the ability of various centromeric repeat sequences to recruit heterochromatin assembly to an ectopic euchromatic site. 2) Determine whether RNA mediated heterochromatin formation normally occurs in cis or in trans. We will address whether the centromeric repeat sequences located at heterochromatic regions in fission yeast function to recruit heterochromatin assembly in cis or rather act as targets for heterochromatin targeting via silencing signals, such as centromeric small RNAs generated in trans. 3) Identify factors involved in initiation of heterochromatin assembly. In specific aim 3 we will use a combination of strategies to identify novel factors involved in initiation of heterochromatin assembly. It is important that heterochromatin be confined to discrete chromosomal regions since improper targeting of heterochromatin can lead to misregulation of gene expression and onset of human diseases such as cancer. Our studies may lead to the development of techniques that allow manipulation of heterochromatic domains which would be extremely useful not only as experimental tools to study gene function but also as potential treatments for human disease.