Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism

Abstract

Background 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. Results 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. Conclusions 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.

Nuclear blebs are associated with destabilized chromatin packing domains

“…S2 ). With respect to heterochromatin nanodomains in GSK343 treated HCT116 cells[32, 33] and TSA treated U2OS cells, these were smaller than those in control cells and in lamin B-depletion as expected due to the inhibition of heterochromatin enzymes within the nuclear body ( Fig. 4D-F; Fig.…”

“…[2, 34] To assess the impact of lamin degradation on nuclear morphology, we applied the AID system to HCT116 colorectal carcinoma epithelial cells to induce simultaneous degradation of lamin B1 and lamin B2 as previously described. [32, 33, 35] Using immunofluorescence imaging, we quantified the percentages of nuclear blebbing in HCT116 LMN(B1&B2)-AID cells before and after depletion of B-type lamins by auxin treatment for 24 hours. We found that in comparison to untreated controls, auxin treatment promoted a significant increase in the percentage of cells containing nuclear blebs (2.07% vs 6.23%; 4.163 ± 1.033 (Mean difference ± SEM), p-value = 0.016, Student’s t-test) ( Fig.…”

“…Although loss of B-type lamins and inhibition of heterochromatin enzymes both induced nuclear morphological changes and increased FMM within nuclear blebs, it is important to consider that these perturbations may not always reflect the same changes in cell phenotype. While B-type lamins are required for proper spatial positioning of heterochromatin and gene-specific loci[8, 32, 33], B-type lamin loss and heterochromatin disruption may impact different cellular mechanisms that give rise to these morphological changes. For example, in previous work, we found that decreasing heterochromatin promoted decreased nuclear rigidity and increased nuclear blebbing without necessarily altering lamins.…”

“…In addition to quantifying packing domain structure in live cells, PWS microscopy allows measurement of the effective diffusion coefficient, D e , and the fractional moving mass (FMM) which quantifies the fraction of chromatin demonstrating coherent motion within a diffraction limited volume. Utilizing this technique, we have previously demonstrated that B-type lamin depletion is associated with increased levels of chromatin fractional moving mass and repositioning of heterochromatin cores [32, 33].…”

N-terminal tags impair the ability of Lamin A to provide structural support to the nucleus

“…This leaves open the possibility that other N-terminal tags or internal tags exist that could lead to less severe or no effects on lamin function. Several recent studies reported the use of degron tags fused to lamin (Beer et al, 2019; Pujadas Liwag et al, 2024) or inserted internally within the lamin sequence (Liu et al, 2023), without apparent defects in lamin function; these reports, however, did not present data on the mechanical contributions of tagged lamin to nuclear deformability nor included an expression-controlled comparison to wild-type lamin, as performed in this study, leaving unresolved whether the tag negatively affected some lamin functions.…”