Mechanistic dissection of HP1 mediated heterochromatin

描述（由申请人提供）：异染色质的扩散对于遗传性地沉默基因组的大部分区域至关重要，因此在发育过程中产生和维持细胞身份。异染色质扩散缺失导致的基因异常激活与浸润性乳腺癌密切相关，而异染色质异常扩散导致的基因异常沉默与髓性白血病密切相关。除了基因沉默外，异染色质在重组和染色体分离中起着至关重要的作用。在最保守的异染色质形式的核心是HP1蛋白和组蛋白H3 （H3K9me3）赖氨酸9甲基化的染色质之间形成的复合物。HP1蛋白在异染色质功能中具有以下关键作用：(1)假设HP1-核小体复合体将效应分子招募到H3K9me3染色质上。矛盾的是，既能促进也能限制异染色质进一步扩散的效应物被招募。这些对立活动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HP1蛋白通过跨多个核小体的寡聚化直接介导异染色质的扩散。(3)假设HP1蛋白浓缩染色质，从而直接减少DNA进入转录机制。尽管这些特性在异染色质的体内功能中处于中心地位，但HP1如何完成这些作用的分子基础尚不清楚。我们的新发现是，S. pombe HP1的主要异构体Swi6，以一种依赖于H3K9me3的识别和核小体的附加特征的方式，从自动抑制状态切换到传播能力状态。我们将在这些和其他结果的基础上解决S. pombe模型系统中的以下问题：(i) HP1蛋白如何与不同的效应物相互作用，（iii）为什么不同的HP1同种异构体具有不同的功能，（iii） HP1蛋白如何在染色质上扩散，以及（iii） HP1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结合。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.