Department of Biophysics, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Biology, Brandeis University, Waltham, MA 02451, USA.
Nucleic Acids Res. 2024 Oct 14;52(18):10731-10746. doi: 10.1093/nar/gkae692.
Visualizing and measuring molecular-scale interactions in living cells represents a major challenge, but recent advances in single-molecule super-resolution microscopy are bringing us closer to achieving this goal. Single-molecule super-resolution microscopy enables high-resolution and sensitive imaging of the positions and movement of molecules in living cells. HP1 proteins are important regulators of gene expression because they selectively bind and recognize H3K9 methylated (H3K9me) histones to form heterochromatin-associated protein complexes that silence gene expression, but several important mechanistic details of this process remain unexplored. Here, we extended live-cell single-molecule tracking studies in fission yeast to determine how HP1 proteins interact with their binding partners in the nucleus. We measured how genetic perturbations that affect H3K9me alter the diffusive properties of HP1 proteins and their binding partners, and we inferred their most likely interaction sites. Our results demonstrate that H3K9 methylation spatially restricts HP1 proteins and their interactors, thereby promoting ternary complex formation on chromatin while simultaneously suppressing off-chromatin binding. As opposed to being an inert platform to direct HP1 binding, our studies propose a novel function for H3K9me in promoting ternary complex formation by enhancing the specificity and stimulating the assembly of HP1-protein complexes in living cells.
在活细胞中可视化和测量分子尺度的相互作用是一个主要的挑战，但单分子超分辨率显微镜的最新进展使我们更接近实现这一目标。单分子超分辨率显微镜能够高分辨率和敏感地成像活细胞中分子的位置和运动。HP1 蛋白是基因表达的重要调节剂，因为它们选择性地结合并识别 H3K9 甲基化（H3K9me）组蛋白，形成沉默基因表达的异染色质相关蛋白复合物，但这个过程的几个重要机制细节仍未被探索。在这里，我们将裂殖酵母的活细胞单分子跟踪研究扩展到确定 HP1 蛋白如何与其核内结合伙伴相互作用。我们测量了影响 H3K9me 的遗传扰动如何改变 HP1 蛋白及其结合伙伴的扩散性质，并推断了它们最可能的相互作用位点。我们的结果表明，H3K9 甲基化在空间上限制了 HP1 蛋白及其相互作用因子，从而促进了染色质上的三元复合物形成，同时抑制了染色质外的结合。与作为指导 HP1 结合的惰性平台相反，我们的研究提出了 H3K9me 的一个新功能，通过增强特异性和刺激 HP1 蛋白复合物在活细胞中的组装，促进三元复合物的形成。
