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.
| Original language | English (US) |
|---|---|
| Article number | 77 |
| Journal | Genome biology |
| Volume | 25 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2024 |
Funding
This work was supported by NSF grants EFMA-1830961 and EFMA-1830969 and NIH grants R01CA228272, U54 CA268084, and U54 CA261694. It was also supported by JSPS KAKENHI grant (JP21H0419) and 5 UM1 HG012649. L.A. was supported by the NIH Training Grant T32AI083216. Philanthropic support was generously received from Rob and Kristin Goldman, the Christina Carinato Charitable Foundation, Mark E. Holliday and Mrs. Ingeborg Schneider, and Mr. David Sachs. E.L.A. was supported by the Welch Foundation (Q-1866), a McNair Medical Institute Scholar Award, an NIH Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375), a US-Israel Binational Science Foundation Award (2019276), the Behavioral Plasticity Research Institute (NSF DBI-2021795), NSF Physics Frontiers Center Award (NSF PHY-2019745), and the National Human Genome Research Institute of the National Institutes of Health under award number RM1HG011016-01A1. KLM was supported by the Hyundai Hope on Wheels Hope Scholar Grant, an Early Investigator grant from CURE Childhood Cancer, Unravel Pediatric Cancer, Stanley Manne Children\u2019s Research Institute, and Ann and Robert H. Lurie Children\u2019s Hospital of Chicago under the Molecular and Translational Cancer Biology Neighborhood. Computational analysis of Hi-C data was supported in part through the computational resources and staff contributions provided by the Genomics Compute Cluster, which is jointly supported by the Feinberg School of Medicine, the Center for Genetic Medicine, and Feinberg\u2019s Department of Biochemistry and Molecular Genetics, the Office of the Provost, the Office for Research, and Northwestern Information Technology. The Genomics Compute Cluster is part of Quest, Northwestern University\u2019s high-performance computing facility, with the purpose to advance research in genomics. This work was supported by the Northwestern University \u2013 Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). Flow Cytometry Cell Sorting was performed in collaboration with Paul Mehl on a BD FACSAria SORP system and BD FACSymphony S6 SORP system, purchased through the support of NIH 1S10OD011996-01 and 1S10OD026814-01. This research was supported by the National Cancer Institute P30-CA044579 Center Grant (University of Virginia Flow Cytometry Core, RRID: SCR_017829). RNA sequencing services were performed at The Genome Analysis and Technology Core (RRID:SCR_018883).
Keywords
- 3D chromatin organization
- Auxin-inducible degron system
- CRISPR-Sirius; in situ Hi-C
- Lamin-associated domains
- Nuclear lamina
- Partial Wave Spectroscopic Microscopy
- Topologically associated domains
ASJC Scopus subject areas
- Ecology, Evolution, Behavior and Systematics
- Genetics
- Cell Biology