背景:B型层粘连蛋白是与三维基因组结构相互作用的关键核包膜蛋白。然而,确定B-层粘连蛋白在动态基因组组织中的直接作用一直具有挑战性,因为它们的联合消耗严重影响细胞活力。为了克服这一点,我们使用生长素诱导的Degron技术对哺乳动物细胞进行工程改造,使其能够快速,完全降解内源性B型层板蛋白。
结果:使用活细胞双部分波光谱(双PWS)显微镜,随机光学重建显微镜(STORM),就地Hi-C,CRISPR-Sirius,和荧光原位杂交(FISH),我们证明,层粘连蛋白B1和层粘连蛋白B2是核外周的关键结构成分,它们为外周相关基因创造了抑制区室。LaminB1和LaminB2耗竭最低限度地改变高阶染色质折叠,但破坏细胞形态,显著增加染色质迁移率,重新分配组成型和兼性异染色质,并在层粘连蛋白相关域(LAD)边界内和附近诱导差异基因表达。严重的,我们证明,随着LAD内上调的基因径向向内移动,染色质区域会扩展。我们的结果表明,B型层粘连蛋白的作用机制来自它们在限制染色质运动和基因特异性位点的空间定位中的作用。异染色质,和染色质结构域。
结论:我们的研究结果表明,而B型层粘连蛋白降解不会显著改变基因组拓扑结构,它对单细胞水平的三维染色质构象在层相关外周和非LAD相关核内部都有重要意义,伴随着全基因组转录变化.这引发了关于层粘连蛋白B1和层粘连蛋白B2在细胞功能和疾病中的个体和重叠作用的有趣问题。
B-type
lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-
lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type
lamins using Auxin-inducible degron technology.
Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type
lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains.
Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.