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Abstract BackgroundB-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. ResultsUsing 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. ConclusionsOur 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.
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Heterologous expression of Dictyostelium discoideum NE81 in mouse embryo fibroblasts reveals conserved mechanoprotective roles of lamins
Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.
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- Award ID(s):
- 2022048
- PAR ID:
- 10523272
- Editor(s):
- Discher, Dennis
- Publisher / Repository:
- The American Society of Cell Biology
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 35
- Issue:
- 1
- ISSN:
- 1059-1524
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E23-05-0193
