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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. 

Abstract Chromatin organization regulates transcription to influence cellular plasticity and cell fate. We explored whether chromatin nanoscale packing domains are involved in stemness and response to chemotherapy. Using an optical spectroscopic nanosensing technology we show that ovarian cancer‐derived cancer stem cells (CSCs) display upregulation of nanoscale chromatin packing domains compared to non‐CSCs. Cleavage under targets and tagmentation (CUT&Tag) sequencing with antibodies for repressive H3K27me3 and active H3K4me3 and H3K27ac marks mapped chromatin regions associated with differentially expressed genes. More poised genes marked by both H3K4me3 and H3K27me3 were identified in CSCs vs. non‐CSCs, supporting increased transcriptional plasticity of CSCs. Pathways related to Wnt signaling and cytokine‐cytokine receptor interaction were repressed in non‐CSCs, while retinol metabolism and antioxidant response were activated in CSCs. Comparative transcriptomic analyses showed higher intercellular transcriptional heterogeneity at baseline in CSCs. In response to cisplatin, genes with low baseline expression levels underwent the highest upregulation in CSCs, demonstrating transcriptional plasticity under stress. Epigenome targeting drugs downregulated chromatin packing domains and promoted cellular differentiation. A disruptor of telomeric silencing 1‐like (Dot1L) inhibitor blocked transcriptional plasticity, reversing stemness. These findings support that CSCs harbor upregulated chromatin packing domains, contributing to transcriptional and cell plasticity that epigenome modifiers can target.