Targeting of Heterochromatin Assembly in Schizosaccharomyces pombe

描述（由申请人提供）：异染色质参与了广泛的细胞过程的调控，包括基因表达、染色体分离和细胞分化。然而，将异染色质定位到基因组的特定区域所涉及的机制尚不完全清楚。当我们发现由异染色质着丝粒序列合成的双链RNA被RNAi机制快速加工导致异染色质序列特异性靶向着丝粒时，我们对这个问题有了深入的了解。这个建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目标。我们的具体目标是：1)确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，哪些着丝粒重复序列对异染色质组装很重要，以及它们如何发挥招募沉默的作用尚不清楚。我们将通过分析各种着丝粒重复序列将异染色质组装募集到异位常染色质位点的能力来直接验证这一点。2)确定RNA介导的异染色质形成是正常发生在顺式还是反式。我们将探讨裂变酵母中位于异染色质区域的着丝粒重复序列是否以顺式方式招募异染色质组装，或者通过沉默信号（如反式中产生的着丝粒小rna）作为异染色质靶向的靶标。3)确定参与异染色质组装起始的因素。在具体目标3中，我们将使用多种策略组合来确定参与异染色质组装起始的新因素。重要的是，异染色质被限制在离散的染色体区域，因为异染色质的不当靶向可能导致基因表达的失调和人类疾病（如癌症）的发病。我们的研究可能会导致技术的发展，允许操作异色结构域，这将是非常有用的，不仅是研究基因功能的实验工具，而且是潜在的治疗人类疾病。

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.