Mechanistic dissection of HP1 mediated heterochromatin

描述（由申请人提供）：异染色质的扩散对于基因组的大区域遗传沉默至关重要，因此对于发育期间产生和维持细胞身份至关重要。异染色质扩散丢失导致的不合理基因激活与浸润性乳腺癌密切相关，而异染色质扩散异常导致的不合理基因沉默与髓性白血病密切相关。除了基因沉默，异染色质在重组和染色体分离中起着至关重要的作用。在异染色质的最保守形式的核心是HP 1蛋白和组蛋白H3的赖氨酸9（H3 K9 me 3）上甲基化的染色质之间形成的复合物。HP 1蛋白在异染色质功能中具有以下关键作用：（1）假设HP 1-核小体复合物将效应分子募集到H3 K9 me 3染色质。巧合的是，激活和限制异染色质进一步扩散的效应子被募集。这些相反的活性之间的平衡被认为决定了组装的异染色质的功能和稳定性。(2)假设HP 1蛋白通过跨多个核小体寡聚化直接介导异染色质的扩散。(3)假设HP 1蛋白质浓缩染色质，从而直接减少DNA对转录机器的访问。尽管这些特性对异染色质的体内功能至关重要，但对HP 1如何完成这些作用的分子基础知之甚少。我们发现，主要的S.粟酒裂殖酵母HP 1亚型，从一个自动抑制状态转换到一个扩展能力状态的方式，这取决于识别H3 K9 me 3和核小体的其他功能。我们将建立在这些和其他结果，以解决以下问题在S。pombe模型系统：（i）HP 1蛋白如何与不同的效应物相互作用，（iii）为什么不同的HP 1亚型具有不同的功能，（iii）HP 1蛋白如何在染色质中传播，（iii）HP 1组装对染色质结构有什么影响？我们将使用定量生化方法和尖端电子冷冻显微镜方法的组合。我们还将在体内测试我们模型的关键预测。

DESCRIPTION (provided by applicant): The spread of heterochromatin is crucial for heritably silencing large regions of the genome and consequently for generating and maintaining cell identity during development. Illegitimate gene activation from loss of heterochromatin spread is strongly linked to invasive breast cancers while illegitimate gene silencing from aberrant heterochromatin spread is strongly linked to myeloid leukemias. In addition to gene silencing, heterochromatin plays crucial roles in recombination and chromosome segregation. At the core of the most conserved form of heterochromatin is the complex formed between HP1 proteins and chromatin methylated on lysine 9 of histone H3 (H3K9me3). The following key roles have been attributed to HP1 proteins in heterochromatin function: (1) The HP1-nucleosome complex is hypothesized to recruit effector molecules to H3K9me3 chromatin. Paradoxically, effectors that both, enable as well as restrict further heterochromatin spread are recruited. The balance between these opposing activities is thought to dictate the functions and stability of the assembled heterochromatin. (2) HP1 proteins are hypothesized to directly mediate the spread of heterochromatin by oligomerizing across multiple nucleosomes. (3) HP1 proteins are hypothesized to condense chromatin and thereby directly reduce the access of DNA to the transcription machinery. Despite the centrality of these properties to the in vivo functions of heterochromatin, the molecular basis for how HP1 accomplishes these roles is poorly understood. We have made the new discovery that Swi6, the major S. pombe HP1 isoform, switches from an auto-inhibited state to a spreading-competent state in a manner that depends on recognition of H3K9me3 and additional features of a nucleosome. We will build on these and additional results to address the following questions in the S. pombe model system: (i) how do HP1 proteins interact with different effectors, (iii) why do different HP1 isoforms have different functions, (iii) how do HP1 proteins spread across chromatin, and (iii) what does HP1 assembly do to chromatin structure? We will use a combination of quantitative biochemical methods and cutting edge electron cryo-microscopy approaches. We will also test key predictions of our models in vivo.