Biochemical dissection of epigenetic inheritance by Polycomb group proteins

描述(由申请人提供)：表观遗传机制通过在细胞周期中维持基因调控信息，使基因表达模式稳定。在某些情况下，这被认为是通过可遗传的染色质结构改变而发生的。由果蝇多梳组(PcG)基因编码的蛋白质在发育过程中维持基因表达的关键模式。这些基因及其功能在哺乳动物中是保守的，其中PcG。蛋白质也与许多癌症有关。为了了解这些蛋白质如何利用染色质结构来维持基因沉默，并开始重建表观遗传过程中的步骤，将对Polycomb组蛋白和染色质进行体外生化研究。首先，将对关键的PcG蛋白--后性梳子(PSC)进行详细研究，以确定该蛋白如何改变染色质构象。然后将对破坏PSC体外活性的突变进行体内功能测试。其次，PcG介导的调节可以非常稳定。为了验证这种稳定性反映了PcG蛋白与染色质之间稳定相互作用的假设，我们将在体外测量不同条件下PcG与染色质的复合体的脱落率。最后，一个尚未解开的重大谜团是，表观遗传调节因子，如PcG蛋白，如何通过细胞周期的破坏性事件来维持基因调控信息。为了开始通过DNA复制重建表观遗传信息通道，将在体外复制PcG蛋白结合的染色质，并决定PcG蛋白-染色质相互作用的命运。为了将这些生化结果与体内现象联系起来，将确定PcG蛋白通过S相与染色质的联系，以及DNA复制在体内PcG蛋白耗尽时失去抑制中的作用。与公共健康相关：表观遗传机制通过使基因表达模式稳定，帮助通过发育保持细胞身份。它们还可以锁定异常的调控事件，如那些触发细胞转化和癌症的事件。多梳蛋白被认为是发育的表观遗传调节器，在许多癌症中也上调。这项研究旨在了解这些蛋白质是如何发挥作用的，这将有助于了解它们在癌症中的作用，并最终可能使针对它们的治疗目的成为可能。

DESCRIPTION (provided by applicant): Epigenetic mechanisms make gene expression patterns stable by maintaining gene regulatory information through the cell cycle. In some cases, this is thought to occur through heritable modification of chromatin structure. Proteins encoded by the Drosophila Polycomb Group (PcG) genes maintain key patterns of gene expression during development. These genes and their functions are conserved in mammals where PcG. proteins are also linked to many cancers. To understand how these proteins use chromatin structure to maintain gene silencing and to begin to reconstitute the steps in epigenetic inheritance, in vitro biochemical studies of Polycomb group proteins and chromatin will be carried out. First, detailed studies of a key PcG protein, Posterior Sex Combs (PSC), will be carried out to determine how this protein alters chromatin conformation. Mutations which disrupt in vitro activities of PSC will then be tested for in vivo function. Second, PcG-mediated regulation can be very stable. To test the hypothesis that this stability reflects stable interactions between PcG proteins and chromatin, the off rate of PcG complexes from chromatin will be measured under various conditions in vitro. Finally, a major unsolved mystery is how epigenetic regulators such as PcG proteins maintain gene regulatory information through the disruptive events of the cell cycle. To begin to reconstitute passage of epigenetic information through DNA replication, PcG protein-bound chromatin will be replicated in vitro and the fate of PcG protein-chromatin interactions determined. To connect these biochemical results with in vivo phenomena, the association of PcG proteins with chromatin through S-phase and role of DNA replication in the loss of repression upon depletion of PcG proteins in vivo will be determined. Relevance to public health: Epigenetic mechanisms help maintain cell identities through development by making patterns of gene expression stable. They can also lock aberrant regulatory events, such as those that trigger cellular transformation and cancer, into place. Polycomb proteins are thought to be epigenetic regulators of development and are also upregulated in many cancers. This study aims to understand how these proteins function, which will help understand their role in cancer and may eventually make it possible to target them for therapeutic purposes.