Lamin A/C modulates spatial organization and function of the Hsp70 gene locus via nuclear myosin I.

The structure-function relationship of the nucleus is tightly regulated, especially during heat shock. Typically, heat shock activates molecular chaperones that prevent protein misfolding and preserve genome integrity. However, the molecular mechanisms that regulate nuclear structure-function relationships during heat shock remain unclear. Here, we show that lamin A and C (hereafter lamin A/C; both lamin A and C are encoded by LMNA) are required for heat-shock-mediated transcriptional induction of the Hsp70 gene locus (HSPA genes). Interestingly, lamin A/C regulates redistribution of nuclear myosin I (NM1) into the nucleus upon heat shock, and depletion of either lamin A/C or NM1 abrogates heat-shock-induced repositioning of Hsp70 gene locus away from the nuclear envelope. Lamins and NM1 also regulate spatial positioning of the SC35 (also known as SRSF2) speckles - important nuclear landmarks that modulates Hsp70 gene locus expression upon heat shock. This suggests an intricate crosstalk between nuclear lamins, NM1 and SC35 organization in modulating transcriptional responses of the Hsp70 gene locus during heat shock. Taken together, this study unravels a novel role for lamin A/C in the regulation of the spatial dynamics and function of the Hsp70 gene locus upon heat shock, via the nuclear motor protein NM1.This article has an associated First Person interview with the first author of the paper.

Imaging Chromosome Territory and Gene Loci Positions in Cells Grown on Soft Matrices.

It is well established that the genome is non-randomly organized in the interphase nucleus with gene rich chromosome territories toward the nuclear interior, while gene poor chromosome territories are proximal to the nuclear periphery. In vivo tissue stiffness and architecture modulates cell type-specific genome organization and gene expression programs. However, the impact of external mechanical forces on the non-random organization of the genome is not completely understood. Here we describe a modified protocol for visualizing chromosome territories and gene loci positions in cells exposed to reduced matrix stiffness by employing soft polyacrylamide matrices. 3-Dimensional Fluorescence In Situ Hybridization (3D-FISH) protocol followed by image analyses performed on cells exposed to extracellular matrices of varying stiffness properties, enables the determination of the dynamics of chromosome territories as well as gene loci in the interphase nucleus. This will be useful in understanding how chromosome territories respond to changes in substrate stiffness and the potential correlation between the repositioning of chromosome territories and their respective transcriptional profiles.

Lamin A/C and Emerin depletion impacts chromatin organization and dynamics in the interphase nucleus.

BACKGROUND: Nuclear lamins are type V intermediate filament proteins that maintain nuclear structure and function. Furthermore, Emerin - an interactor of Lamin A/C, facilitates crosstalk between the cytoskeleton and the nucleus as it also interacts with actin and Nuclear Myosin 1 (NM1). RESULTS: Here we show that the depletion of Lamin A/C or Emerin, alters the localization of the nuclear motor protein - Nuclear Myosin 1 (NM1) that manifests as an increase in NM1 foci in the nucleus and are rescued to basal levels upon the combined knockdown of Lamin A/C and Emerin. Furthermore, Lamin A/C-Emerin co-depletion destabilizes cytoskeletal organization as it increases actin stress fibers. This further impinges on nuclear organization, as it enhances chromatin mobility more toward the nuclear interior in Lamin A/C-Emerin co-depleted cells. This enhanced chromatin mobility was restored to basal levels either upon inhibition of Nuclear Myosin 1 (NM1) activity or actin depolymerization. In addition, the combined loss of Lamin A/C and Emerin alters the otherwise highly conserved spatial positions of chromosome territories. Furthermore, knockdown of Lamin A/C or Lamin A/C-Emerin combined, deregulates expression levels of a candidate subset of genes. Amongst these genes, both KLK10 (Chr.19, Lamina Associated Domain (LAD+)) and MADH2 (Chr.18, LAD-) were significantly repressed, while BCL2L12 (Chr.19, LAD-) is de-repressed. These genes differentially reposition with respect to the nuclear envelope. CONCLUSIONS: Taken together, these studies underscore a remarkable interplay between Lamin A/C and Emerin in modulating cytoskeletal organization of actin and NM1 that impinges on chromatin dynamics and function in the interphase nucleus.

Emerin modulates spatial organization of chromosome territories in cells on softer matrices.

Cells perceive and relay external mechanical forces into the nucleus through the nuclear envelope. Here we examined the effect of lowering substrate stiffness as a paradigm to address the impact of altered mechanical forces on nuclear structure-function relationships. RNA sequencing of cells on softer matrices revealed significant transcriptional imbalances, predominantly in chromatin associated processes and transcriptional deregulation of human Chromosome 1. Furthermore, 3-Dimensional fluorescence in situ hybridization (3D-FISH) analyses showed a significant mislocalization of Chromosome 1 and 19 Territories (CT) into the nuclear interior, consistent with their transcriptional deregulation. However, CT18 with relatively lower transcriptional dysregulation, also mislocalized into the nuclear interior. Furthermore, nuclear Lamins that regulate chromosome positioning, were mislocalized into the nuclear interior in response to lowered matrix stiffness. Notably, Lamin B2 overexpression retained CT18 near the nuclear periphery in cells on softer matrices. While, cells on softer matrices also activated emerin phosphorylation at a novel Tyr99 residue, the inhibition of which in a phospho-deficient mutant (emerinY99F), selectively retained chromosome 18 and 19 but not chromosome 1 territories at their conserved nuclear locations. Taken together, emerin functions as a key mechanosensor, that modulates the spatial organization of chromosome territories in the interphase nucleus.