Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes
Quantitative FLIM-FRET Microscopy to Monitor Nanoscale Chromatin Compaction In Vivo Reveals Structural Roles of Condensin Complexes
复制标题
DOI：
10.1016/j.celrep.2017.01.043
复制
发表时间：
2017-02-14
期刊：
影响因子：
8.8
通讯作者：
Feil, Robert
中科院分区：
文献类型：
--
作者：
Lleres, David;Bailly, Aymeric P.;Feil, Robert
在活细胞和组织中，后生动物的基因组是如何在纳米尺度上构建的仍然未知。在这里，我们采用了定量FRET（福斯特共振能量转移）为基础的荧光寿命成像显微镜（FLIM）的方法来测定纳米级染色质压实在活的生物体。选择秀丽隐杆线虫作为模型系统。通过测量组蛋白标记的荧光蛋白之间的FRET，我们可视化了不同的染色体区域，并量化了减数分裂细胞中不同水平的纳米级压实。利用RNAi和重复染色体外阵列方法，我们确定了异染色质状态，并发现其结构在体内呈现由异染色质蛋白-1（HP1）和SETDB1 h3 -赖氨酸-9甲基转移酶同源物控制的纳米级致密组织。接下来，我们对凝缩蛋白复合物进行了功能探索。我们发现凝缩蛋白I和凝缩蛋白II对异染色质压实至关重要，凝缩蛋白I还控制低压实区域。我们的数据表明，在活体动物中，纳米级染色质压实不仅由组蛋白修饰剂和读取器控制，还由凝聚蛋白复合物控制。
How metazoan genomes are structured at the nanoscale in living cells and tissues remains unknown. Here, we adapted a quantitative FRET (Forster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanoscale chromatin compaction in living organisms. Caenorhabditis elegans was chosen as a model system. By measuring FRET betweenhistone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1(HP1) and SETDB1 H3-lysine-9 methyltransferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.