Biophysical interrogation of the RNA-mediated mechanisms regulating Polycomb Repressive Complex 2 activity

项目总结 多梳抑制复合体2(PRC2)是一种组蛋白甲基转移酶(HMTase)，它顺序地沉积三个 组蛋白H3的赖氨酸27(H3K27me1/2/3)上的甲基，其活性对表观遗传沉默是至关重要的。 发展和癌症。鉴于PRC2的关键功能和功能，人们对PrC2如何针对遗传位点非常感兴趣 靶基因的丰富性。众所周知，PRC2与RNA广泛相互作用，这些相互作用调节 PrC2‘S定位于表观遗传沉默的基因组位点。然而，关于自然和自然的细节 PRC2的S受RNA调控的机制(S)仍存在较大争议。虽然一些研究提出了一个角色，即 核糖核酸在PrC2的S向染色质募集中的作用，更多的人提出了在PrC2从染色质中驱逐和/或抑制的作用 PRC2的活动。值得注意的是，最近的一项工作表明，PRC2-RNA相互作用在体内对 维持H3K27me3水平和PrC2靶基因的染色质占有率。因此，尽管有证据表明RNA扮演着 在某些条件下，它在将PRC2从染色质中驱逐出去的作用，似乎也必须促进染色质结合和 H3K27me3至少在某些情况下沉积。到目前为止，PRC2文献缺乏机制的直接证据 这可以协调这些先前的数据，并解释RNA介导的染色质结合和PRC2的HMTase活性。我的 初步数据出人意料地暗示，PrC2具有在多核苷酸之间直接转移的内在能力，而这 这一现象可能广泛地与RNA介导的染色质修饰酶的调节有关。基于这些 和其他数据，我假设了一个“移交”模型，在这个模型中，PRC2可以直接在新生RNA和 空间近端染色质，以促进H3K27me3的沉积或从染色质中驱逐。在此提出的研究将 定量pRC2‘S与核糖核酸和核小体的结合和竞争动力学，确定多核苷酸物种 PRC2可以转移，表征这种现象在其他物种中的流行情况和生物物理要求 蛋白质，并探讨核小体与核小体的空间关系对pRC2‘S HMTase活性的影响。调查结果来自 这些研究将扩大我们对染色质修饰酶是如何调节的理解，这可能 这对我们理解细胞分化、胚胎发育和癌症有着深远的意义。

PROJECT SUMMARY Polycomb repressive complex 2 (PRC2) is a histone methyltransferase (HMTase) that sequentially deposits three methyl groups onto lysine 27 of histone H3 (H3K27me1/2/3), and its activity is crucial for epigenetic silencing during development and cancer. How PRC2 is targeted to genetic loci is of considerable interest, given its critical function and abundance of target genes. It’s well accepted that PRC2 interacts broadly with RNA, and that these interactions regulate PRC2’s localization at genomic sites destined for epigenetic silencing. However, the details about the nature and mechanism(s) of PRC2’s regulation by RNA remain quite controversial. While some studies have proposed a role for RNA in PRC2’s recruitment to chromatin, more have suggested roles in PRC2 eviction from chromatin and/or inhibition of PRC2 activity. Notably, a recent work has demonstrated that the PRC2-RNA interaction is critical in vivo for maintaining H3K27me3 levels and chromatin occupancy at PRC2 target genes. Thus, while there’s evidence RNA plays a role in evicting PRC2 from chromatin under certain conditions, it also seems it must facilitate chromatin binding and H3K27me3 deposition in at least some circumstances. To date, the PRC2 literature lacks direct evidence for a mechanism that can reconcile these prior data and explain RNA-mediated chromatin-binding and HMTase activity by PRC2. My preliminary data unexpectedly imply PRC2 has the intrinsic capacity to transfer directly between polynucleotides, and this phenomenon could be broadly relevant to RNA-mediated regulation of chromatin-modifying enzymes. Based on these and other data, I hypothesize a ‘hand-off’ model where PRC2 can be directly transferred between nascent RNA and spatially proximal chromatin to facilitate H3K27me3 deposition or eviction from chromatin. Studies proposed herein will quantify PRC2’s binding and competition kinetics for RNA and DNA/nucleosomes, determine the polynucleotide species PRC2 could be transferred between, characterize the prevalence and biophysical requisites of this phenomenon in other proteins, and interrogate the effect of RNA-nucleosome spatial relationships on PRC2’s HMTase activity. Findings from these studies will expand our understanding of how chromatin-modifying enzymes are regulated, which could have profound implications in our understanding of cell differentiation, embryonic development, and cancer.