Characterization of open chromatin sensitive to actin polymerization using DSP-MNase-DNA-seq
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE257539 | GEO | 2024/08/28
REPOSITORIES: GEO
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Project description:The trans-luminal LINC (Linker of Nucleoskeleton and Cytoskeleton) complex plays a central role in nuclear mechanotransduction by coupling the nucleus with cytoskeleton. High spatial density and active dynamics of LINC complex have hindered its precise characterization for the understanding of underlying mechanisms how the linkages sense and respond to mechanical stimuli. In this study, we focus on SUN2, a core component of LINC complex interconnecting the nuclear lamina and actin cytoskeleton and apply single molecule super-resolution imaging to reveal how SUN2 responds to actomyosin contractility. Using stochastic optical reconstruction microscopy (STORM), we quantitated the distribution pattern and density of SUN2 on the basal nuclear membrane. We found that SUN2 undergoes bidirectional translocation between ER and nuclear membrane in response to actomyosin contractility, suggesting that dynamic constrained force on SUN2 is required for its proper distribution. Furthermore, single molecule imaging unveils interesting dynamics of SUN2 molecules that are regulated by both actomyosin contractility and laminA/C network, whereas SUN2 oligomeric states are not affected by actomyosin contractility. Lastly, the mechanical response of SUN2 to actomyosin contractility was found to regulate expression of mechano-sensitive genes located in lamina-associated domains (LADs) and perinuclear heterochromatin. Taken together, our results reveal how SUN2 responds to mechanical cues at the single-molecule level, providing new insights into the mechanism of nuclear mechanotransduction.
2022-10-12 | GSE214854 | GEO
Project description:Desmosomes are specialized cell-cell junctions critically important in the heart, where their disruption promotes arrhythmogenic cardiomyopathy (ACM). Individuals carrying truncating variants in the desmosomal gene desmoplakin (DSP) often show a prominent inflammatory component in the absence of known antigenic stimuli, termed “hot phase” arrythmia. To assess the role of innate immune activation in DSP-cardiomyopathy, we generated engineered heart tissues (EHTs) from two patients with heterozygous DSP truncation variants (DSPtv, p.E1597X and p.R1951X). We also generated EHTs with homozygous DSP deletion (DSP-/-) for modeling. DSP-/- EHTs revealed impaired force production and reduced conduction velocity compared to isogenic controls, features consistent with ACM. RNA sequencing and protein characterization demonstrated a dramatic increase in innate immune activation in DSP-/- EHTs. Similarly, patient-derived DSPtv EHTs show enhanced sensitivity to inflammatory stimuli seen as reduced contractility and greater cytokine release. Inhibition of NFκB signaling improved baseline force production and mechanical response to strain in DSPtv EHTs, indicating that anti-inflammatory modulation may improve mechanical function in DSP deficient hearts. Furthermore, genomic correction of p.R1951X using adenine base editing reduced inflammatory biomarkers in cultured EHTs. Taken together, these data identify that DSPtv promote innate immune activation, creating an arrhythmogenic substrate in the concealed phase of ACM.
2024-07-03 | GSE248187 | GEO
Project description:Arterial endothelial cells (ECs) have the ability to respond to mechanical forces exerted by laminar fluid shear stress. This response is of importance, as it is protective against vascular diseases such as atherosclerosis and aortic aneurysms. Mechanical forces are transmitted at the sites of adhesion to the basal membrane as well as cell-cell junctions where protein complexes connect to the cellular cytoskeleton to relay force into the cell. Here we present a novel protein complex that connects junctional VE-cadherin and radial actin filaments to the LINC complex in the nuclear membrane. We show that the scaffold protein AmotL2 is essential for the formation of radial actin filaments and the flow-induced alignment of aortic endothelial cells. The deletion of endothelial AmotL2 alters nuclear shape as well as subcellular positioning. Molecular analysis shows that VE-cadherin is mechanically associated with the nuclear membrane via binding to AmotL2 and Actin. Furthermore, the deletion of AmotL2 in ECs provoked a pro-inflammatory response and abdominal aortic aneurysms (AAA) in the aorta of mice on a normal diet. Remarkably, transcriptome analysis of AAA samples from human patients revealed a negative correlation between AmotL2 expression and inflammation of the aortic intima. These findings provide a conceptual framework regarding how mechanotransduction in the junctions is coupled with vascular diseases.
2023-08-21 | PXD042661 | Pride
Project description:Arterial endothelial cells (ECs) have the ability to respond to mechanical forces exerted by laminar fluid shear stress. This response is of importance, as it is protective against vascular diseases such as atherosclerosis and aortic aneurysms. Mechanical forces are transmitted at the sites of adhesion to the basal membrane as well as cell-cell junctions where protein complexes connect to the cellular cytoskeleton to relay force into the cell. Here we present a novel protein complex that connects junctional VE-cadherin and radial actin filaments to the LINC complex in the nuclear membrane. We show that the scaffold protein AmotL2 is essential for the formation of radial actin filaments and the flow-induced alignment of aortic endothelial cells. The deletion of endothelial AmotL2 alters nuclear shape as well as subcellular positioning. Molecular analysis shows that VE-cadherin is mechanically associated with the nuclear membrane via binding to AmotL2 and Actin. Furthermore, the deletion of AmotL2 in ECs provoked a pro-inflammatory response and abdominal aortic aneurysms (AAA) in the aorta of mice on a normal diet. Remarkably, transcriptome analysis of AAA samples from human patients revealed a negative correlation between AmotL2 expression and inflammation of the aortic intima. These findings provide a conceptual framework regarding how mechanotransduction in the junctions is coupled with vascular diseases.
2024-10-17 | PXD039256 | Pride
Project description:Mechanical cues influence the shape, growth, and function of tissues and organs and are necessary for the development of engineered tissues. Yet, how cells sense mechanical cues and transduce them into changes in gene expression is not well understood. It is known that mechanical forces transmitted to the nucleus induce chromatin remodeling, promote DNA repair, contribute to the motion of intranuclear organelles and cause direct dissociation of protein complexes inside nuclei. Yet, the extent to which such signals impact gene expression is not understood. Because mechanical forces from the cytoskeleton to the nucleus interior are transmitted by the LINC (linker of nucleoskeleton-to-cytoskeleton) complex, we disrupted the LINC complex and performed genome wide expression studies using RNA sequencing. LINC disruption altered the expression of hundreds of genes at a genome-wide scale. We asked how LINC disruption affected the mechanosensitivity of individual genes by quantifying fold changes in gene expression on soft and stiff substrates. Remarkably, LINC disruption tended to preserve gene mechanosensitivity, but to reverse its direction. LINC disruption did not cause changes in nuclear shape, nor eliminated nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex regulates mechano-sensing at a genome-wide level, and argue for a distinct mechanism that does not require changes in nuclear morphology.
2017-01-20 | GSE77520 | GEO
Project description:Mechanical cues influence the shape, growth, and function of tissues and organs and are necessary for the development of engineered tissues. Yet, how cells sense mechanical cues and transduce them into changes in gene expression is not well understood. It is known that mechanical forces transmitted to the nucleus induce chromatin remodeling, promote DNA repair, contribute to the motion of intranuclear organelles and cause direct dissociation of protein complexes inside nuclei. Yet, the extent to which such signals impact gene expression is not understood. Because mechanical forces from the cytoskeleton to the nucleus interior are transmitted by the LINC (linker of nucleoskeleton-to-cytoskeleton) complex, we disrupted the LINC complex and performed genome wide expression studies using RNA sequencing. LINC disruption altered the expression of hundreds of genes at a genome-wide scale. We asked how LINC disruption affected the mechanosensitivity of individual genes by quantifying fold changes in gene expression on soft and stiff substrates. Remarkably, LINC disruption tended to preserve gene mechanosensitivity, but to reverse its direction. LINC disruption did not cause changes in nuclear shape, nor eliminated nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex regulates mechano-sensing at a genome-wide level, and argue for a distinct mechanism that does not require changes in nuclear morphology.
2017-01-20 | GSE77472 | GEO
Project description:Mechanosignaling, initiated by the extracellular physical forces and propagated through the intracellular cytoskeletal network, triggers signaling cascades leading to processes such as embryogenesis, tissue maintenance and disease development. While signal transduction by transcription factors occurs downstream of cellular mechanosensing, nothing is known about the cell intrinsic mechanisms that can regulate mechanosignaling. Here we show that transcription factor PREP1 (PKNOX1) regulates the expression of LINC complex proteins and mechanotransduction of YAP-TAZ. PREP1 depletion upsets the nuclear membrane protein stoichiometry rendering nuclei soft. However, reduced nuclear tension is not conveyed to the cytoplasm, since these cells display fortified actomyosin network with bigger focal adhesion complexes resulting in greater traction forces at the substratum. Intriguingly, YAP-TAZ remains localized to the cytoplasm irrespective of the substrate rigidity indicating disrupted mechanotransduction. Our data demonstrate a hitherto unknown transcriptional mechanism which regulate mechanosignaling upstream of YAP-TAZ transduction.
2022-03-14 | GSE160286 | GEO
Project description:We developed a new method to extract open-chromatins that contain trans-acting factors (TAFs)-DNA complex crosslinked with dithiobis(succinimidyl propionate) (DSP). Using this method we identified CBFB as a cell density-sensitive TAF, which translocates between the cytoplasm and the nucleus in cell density-dependent manner. Here, we demonstrate that the extracted TAF/DNA complex is also amenable to ChIP-seq analysis.
2023-12-05 | GSE234731 | GEO
Project description:Cell adhesion to the extracellular matrix occurs through integrin-mediated focal adhesions, which sense the mechanical properties of the substrate and impact cellular functions such as cell migration. Mechanotransduction at focal adhesions affects the actomyosin network and contributes to cell migration. Despite being key players in cell adhesion and migration, the role of microtubules in mechanotransduction has been overlooked. Here, we show that substrate rigidity increases microtubule acetylation through β1 integrin signalling in primary rat astrocytes. Moreover, αTAT1, the enzyme responsible for microtubule acetylation, interacts with a major mechanosensing focal adhesion protein, Talin, and is able to tune the distribution of focal adhesions depending on the matrix rigidity. αTAT1 also reorganises the actomyosin network, increases traction force generation and cell migration speed on stiff substrates. Mechanistically, acetylation of microtubules promotes the release of microtubule-associated RhoGEF, GEF-H1 into the cytoplasm, which then leads to RhoA activation and high actomyosin contractility. Thus, we propose a novel feedback loop involving a crosstalk between microtubules and actin in mechanotransduction at focal adhesions whereby, cells sense the rigidity of the substrate through integrin-mediated adhesions, modulate their levels of microtubule acetylation, which then controls the actomyosin cytoskeleton and force transmission on the substrate to promote mechanosensitive cell migration.
2021-11-02 | PXD015871 | Pride