Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types.. Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology. Overall design: Nucleolar and PCH TSA-Seq to map the mean cytological proximity of chromosomes genome-wide relative to nucleoli and pericentric heterochromatin (PCH) in four human cell lines - K562, HFFc6, HCT116, H1-hESC. Different condition were used: 1) Condition C: labeling with 1:3000 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide, 2) Condition E: labeling with 1:300 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide (minor modification: 150ul of Dynabeads M-270 streptavidin (Invitrogen, catalog no. 65306) was used to purify the biotinylated DNA) 3) Condition AI for HFFc6, H1, and K562 and 4) Condition A for HCT116 cells.

间期细胞核内基因组的差异化定位迄今仍鲜有探索。我们对TSA-seq技术进行了拓展与验证，用于绘制四种人类细胞系中核仁及着丝粒周异染色质（pericentric heterochromatin）附近的基因组区域图谱。本研究证实，尺寸更小的染色体更靠近核仁，并进一步解析了该规律：长度处于36~46 Mbp以下的染色体臂呈现出更显著的核仁定向偏好。我们通过不同细胞系中核纤层结合结构域（lamina associated domain, LAD）与核纤层、核仁之间的差异化定位，鉴定出两类LAD子集，这两类子集在所有细胞系中均表现出独特的DNA复制时序与基因表达模式。令人意外的是，本研究在典型的转录抑制性核区域附近发现了具有活性、且与核斑点（nuclear speckle）相关的基因组区域，这一现象可归因于部分细胞类型中核斑点与核仁的紧密邻近关系，以及人类胚胎干细胞（human embryonic stem cells, hESCs）中着丝粒与核斑点的结合关联。本研究借助TSA-seq技术揭示，细胞核内基因组的组织结构比现有模型所提出的更为复杂且具有动态可变性。

整体实验设计：针对四种人类细胞系——K562、HFFc6、HCT116及H1-hESC，采用核仁及着丝粒周异染色质TSA-seq技术，绘制全基因组范围内染色体相对于核仁与着丝粒周异染色质的平均细胞学邻近图谱。本研究设置了四种不同的实验条件：1）条件C：采用1:3000浓度的酪酰胺生物素、50%蔗糖及0.0015%过氧化氢进行标记；2）条件E：采用1:300浓度的酪酰胺生物素、50%蔗糖及0.0015%过氧化氢进行标记（小幅优化：使用150 μL Dynabeads M-270链霉亲和素磁珠（Invitrogen，货号65306）纯化生物素标记的DNA）；3）针对HFFc6、H1及K562细胞系的条件AI；4）针对HCT116细胞系的条件A。