Interactions between the genome and the nuclear lamina are multivalent and cooperative (pA-DamID)

Mammalian genomes contain hundreds of lamina-associated domains (LADs), which are large, often megabase-sized heterochromatin domains that are anchored to the nuclear lamina (NL). Even though LADs play a key role in the spatial organization of the genome and potently repress gene activity, it is not yet understood how their interactions with the NL are encoded in their DNA. Here, we investigated the principles that govern LAD-NL interactions by taking a "LAD scrambling" approach. This approach relies on 1) local "hopping" of a Sleeping Beauty transposon to randomly insert loxP sites within and around a LAD of choice; and 2) Cre-mediated recombination between the loxP sites. This approach is highly efficient, enabling us to establish a large collection of clonal cell lines with deletions and inversions up to ~2Mb in size, spanning LAD and their flanking inter-LAD sequences. Mapping of NL interactions in these clones revealed that a single LAD contacts the NL through multiple regions. These regions act cooperatively and redundantly; however, some have more affinity for the NL and can boost NL contacts of neighbouring sequences. We also observed a crosstalk between two neighbouring LADs, when close enough to each other. Finally, changes in the heterochromatin mark H3K9me3 only partially mirrored changes in NL contacts. They also correlated more with gene expression changes than NL contacts did. These principles of LAD - NL interactions provide insight into the overall architecture of the genome and the impact on gene regulation. Overall design: pA-DamID experiment were performed on parental (non-edited), Cre-recombined or CRISPR/Cas9-edited clones. Lamina association and H3K9me3 deposition were probed using anti-LaminB1 (LB1) (Abcam, ab16048) and anti-H3K9me3 (Diagenone, C15410193) antibodies respectively.

哺乳动物基因组包含数百个核纤层关联结构域（lamina-associated domains, LADs），这类大型、通常达百万碱基对规模的异染色质结构域锚定在核纤层（nuclear lamina, NL）上。尽管LADs在基因组空间构象调控中发挥关键作用，并能强效抑制基因活性，但目前学界仍未明确其与核纤层的相互作用是如何编码于其DNA序列之中的。
本研究采用LAD打乱（LAD scrambling）策略，探究调控LAD与NL相互作用的基本原理。该策略包含两个核心步骤：1）借助睡美人转座子（Sleeping Beauty transposon）的局部跳跃特性，在目标LAD内部及其侧翼区域随机插入loxP位点；2）通过Cre重组酶介导loxP位点间的重组反应。该方法具有极高的实验效率，使我们得以建立大量克隆细胞系，其中涵盖了跨度可达约2Mb的LAD及其侧翼LAD间序列的缺失与倒位突变体。
对这些克隆细胞系中的NL相互作用进行图谱分析后发现，单个LAD可通过多个区域与NL发生接触。这些区域兼具协同性与冗余性；其中部分区域对NL具有更高的结合亲和力，能够增强邻近序列与NL的相互作用。
我们还观察到，当两个相邻LAD间距足够近时，二者之间存在串扰现象。
最后，异染色质标记H3K9me3的变化仅部分反映了NL相互作用的改变，且相较于NL相互作用，其与基因表达变化的相关性更强。
上述LAD-NL相互作用的基本原理，为解析基因组整体架构及其对基因调控的影响提供了新的认知视角。
整体实验设计：本研究对亲本（未编辑）、经Cre重组处理或CRISPR/Cas9编辑的克隆细胞系开展了pA-DamID实验。分别使用抗核纤层蛋白B1（LaminB1, LB1）抗体（Abcam, ab16048）与抗H3K9me3抗体（Diagenone, C15410193），检测核纤层关联状态与H3K9me3的沉积水平。