Heterochromatin protein 1 alpha (HP1α) undergoes a monomer to dimer transition that opens and compacts live cell genome architecture. Heterochromatin protein 1 alpha (HP1α) undergoes a monomer to dimer transition that opens and compacts live cell genome architecture

Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1α). Intriguingly, this super- resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1α protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1α oligomeric state. Specifically, we found HP1α monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1α dimerisation. Collectively, these results demonstrate HP1α to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function. Overall design: To investigate our CV-based quantitation of nuclear wide chromatin density, and identify, on what spatial scales the HP1α monomers and dimers are in opposition, we applied high-throughput chromosome conformation capture (Hi-C) to the chromatin network of HeLa, HeLaKD (H (HP1KD) and HeLaHP1αI165E+KD (KDOE), and quantified the frequency of short to long range chromatin interactions. These three conditions were carried out in duplicate.

我们对活细胞内异染色质纳米结构及其介导基因沉默的能力的认知，长期受限于光学显微镜的衍射极限。为突破这一技术瓶颈，直接解析支撑该致密染色质状态的核小体排布规律，我们将核心组蛋白与核小体间福斯特共振能量转移（Förster resonance energy transfer, FRET）的荧光寿命成像显微镜（fluorescence lifetime imaging microscopy, FLIM）成像技术，与异染色质蛋白1α（heterochromatin protein 1 alpha, HP1α）的分子编辑策略相结合。
有趣的是，结合超分辨纳米级染色质结构表征、荧光波动光谱（fluorescence fluctuation spectroscopy, FFS）以及HP1α蛋白相互作用的FLIM-FRET分析，我们发现核小体排布受HP1α寡聚体状态的差异性调控。具体而言，我们观察到HP1α单体可在全基因组范围内诱导一种此前未被报道的全局核小体间距模式，该模式由组蛋白H3赖氨酸9三甲基化（H3K9me3）介导，且在HP1α发生二聚化后局部减弱。
综上，上述结果表明HP1α可对染色质发挥双重调控作用，拓宽核小体邻近性的动态范围。我们预期该发现将有助于深入理解活细胞异染色质结构如何调控基因组功能。
实验整体设计：为验证基于计算机视觉（Computer Vision, CV）的全细胞核染色质密度定量方法，并明确HP1α单体与二聚体发挥拮抗作用的空间尺度，我们对海拉细胞（HeLa）、海拉HP1敲低细胞（HeLaKD，HP1KD）以及海拉HP1αI165E突变+HP1敲低细胞（HeLaHP1αI165E+KD，KDOE）的染色质网络开展高通量染色体构象捕获（high-throughput chromosome conformation capture, Hi-C）实验，并定量分析短程至长程染色质相互作用的频率。上述三种实验条件均设置生物学重复两次。