ABSTRACT
The p53 family remains a captivating focus of an extensive number of current studies. Accumulating evidence indicates that p53 abnormalities rank among the most prevalent in cancer. Given the numerous existing studies, which mostly focus on the mutations, expression profiles, and functional perturbations exhibited by members of the p53 family across diverse malignancies, this review will concentrate more on less explored facets regarding p53 activation and stabilization by the nuclear pore complex (NPC) in cancer, drawing on several studies. p53 integrates a broad spectrum of signals and is subject to diverse regulatory mechanisms to enact the necessary cellular response. It is widely acknowledged that each stage of p53 regulation, from synthesis to degradation, significantly influences its functionality in executing specific tasks. Over recent decades, a large body of data has established that mechanisms of regulation, closely linked with protein activation and stabilization, involve intricate interactions with various cellular components. These often transcend canonical regulatory pathways. This new knowledge has expanded from the regulation of genes themselves to epigenomics and proteomics, whereby interaction partners increase in number and complexity compared with earlier paradigms. Specifically, studies have recently shown the involvement of the NPC protein in such complex interactions, underscoring the further complexity of p53 regulation. Furthermore, we also discuss therapeutic strategies based on recent developments in this field in combination with established targeted therapies.
Subject(s)
Neoplasms , Nuclear Pore , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Nuclear Pore/metabolism , Nuclear Pore/genetics , Animals , Gene Expression Regulation, NeoplasticABSTRACT
Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.
Subject(s)
DNA Damage , Radiation Tolerance , Transcription Factors , Humans , Transcription Factors/metabolism , DNA/radiation effects , DNA/chemistry , Cell Line, Tumor , Cell Cycle Proteins/metabolism , Enhancer Elements, Genetic , Genome, Human , Nuclear Proteins/metabolism , Bromodomain Containing ProteinsABSTRACT
The size of the nuclear pore should, in principle, prevent HIV-1 entry. However, HIV-1 capsid is able to gain nuclear pore entry. In a recent issue of Nature, Fu et al. and Dickson et al. demonstrate that the HIV-1 capsid mimics the nuclear transport protein karyopherins to access host nuclei.
Subject(s)
HIV Infections , Nuclear Pore , Humans , Capsid/metabolism , Capsid Proteins/metabolism , HIV Infections/metabolism , Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Nuclear Pore Complex Proteins/metabolismABSTRACT
Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.
Subject(s)
Carcinoma, Squamous Cell , Interferon-gamma , Animals , Humans , Mice , B7-H1 Antigen/metabolism , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , CD8-Positive T-Lymphocytes , Cell Line, Tumor , Immunity , Interferon-gamma/metabolism , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/metabolism , Transcription Factors/metabolism , Tumor Microenvironment , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.
Subject(s)
Cell Proliferation , Cryopreservation , Cryoprotective Agents , Polymers , Cryopreservation/methods , Polymers/chemistry , Polymers/pharmacology , Cryoprotective Agents/pharmacology , Cryoprotective Agents/chemistry , Cell Proliferation/drug effects , Animals , Humans , Cell Survival/drug effects , Freezing , MiceABSTRACT
Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.
Subject(s)
Proteins , Single Molecule Imaging , Animals , Mice , Microscopy, Atomic Force/methods , Proteins/chemistry , Cell Nucleus , Brain/diagnostic imaging , MammalsABSTRACT
Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.
ABSTRACT
During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.
ABSTRACT
Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.
Subject(s)
Open Reading Frames , SARS-CoV-2 , Viral Proteins , Amyloid , Peptides , SARS-CoV-2/genetics , Signal Transduction , Viral Proteins/geneticsABSTRACT
Nuclear pore complexes (NPCs) are the central apparatus of nucleocytoplasmic transport. Disease-specific alterations of NPCs contribute to the pathogenesis of many cancers; however, the roles of NPCs in glioblastoma (GBM) are unknown. In this study, we report genomic amplification of NUP107, a component of NPCs, in GBM and show that NUP107 is overexpressed simultaneously with MDM2, a critical E3 ligase that mediates p53 degradation. Depletion of NUP107 inhibits the growth of GBM cell lines through p53 protein stabilization. Mechanistically, NPCs establish a p53 degradation platform via an export pathway coupled with 26S proteasome tethering. NUP107 is the keystone for NPC assembly; the loss of NUP107 affects the integrity of the NPC structure, and thus the proportion of 26S proteasome in the vicinity of nuclear pores significantly decreases. Together, our findings establish roles of NPCs in transport surveillance and provide insights into p53 inactivation in GBM.
Subject(s)
Glioblastoma , Nuclear Pore , Humans , Nuclear Pore/metabolism , Active Transport, Cell Nucleus , Nuclear Pore Complex Proteins/metabolism , Glioblastoma/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.
Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.
Subject(s)
Extracellular Vesicles , Microscopy, Atomic Force/methodsABSTRACT
The maintenance and proliferation of hematopoietic stem cells (HSCs) are tightly regulated by their niches in the bone marrow. The analysis of niche cells or stromal cell lines that can support HSCs has facilitated the finding of novel supporting factors for HSCs. Despite large efforts in the murine bone marrow; however, HSC expansion is still difficult ex vivo, highlighting the need for new approaches to elucidate the molecular elements that regulate HSCs. The zebrafish provides a unique model to study hematopoietic niches as HSCs are maintained in the kidney, allowing for a parallel view of hematopoietic niches over evolution. Here, using a stromal cell line from the zebrafish kidney, zebrafish kidney stromal (ZKS), we uncover that an inhibitor of canonical Wnt signaling, IWR-1-endo, is a potent regulator of HSCs. Coculture assays revealed that ZKS cells were in part supportive of maintenance, but not expansion, of gata2a:GFP+runx1:mCherry+ (gata2a+runx1+) HSCs. Transcriptome analysis revealed that, compared with candidate niche cells in the kidney, ZKS cells weakly expressed HSC maintenance factor genes, thpo and cxcl12, but highly expressed canonical Wnt ligand genes, wnt1, 7bb, and 9a. Thpo supplementation in ZKS culture slightly increased, but inhibition of canonical Wnt signaling by IWR-1-endo treatment largely increased the number of gata2a+runx1+ cells (>2-fold). Moreover, we found that gata2a+runx1+ cells can be maintained by supplementing both IWR-1-endo and Thpo without stromal cells. Collectively, our data provide evidence that IWR-1-endo can be used as a novel supporting factor for HSCs.
Subject(s)
Core Binding Factor Alpha 2 Subunit , Zebrafish , Animals , Cell Proliferation , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Hematopoietic Stem Cells/metabolism , Ligands , Mice , Wnt Signaling Pathway/genetics , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
Epigenetic deregulation plays an essential role in colorectal cancer progression. Bromodomains are epigenetic "readers" of histone acetylation. Bromodomain-containing protein 4 (BRD4) plays a pivotal role in transcriptional regulation and is a feasible drug target in cancer cells. Disease-specific elevation of nucleoporin, a component of the nuclear pore complex (NPC), is a determinant of cancer malignancy, but BRD4-driven changes of NPC composition remain poorly understood. Here, we developed novel aminocyclopropenones and investigated their biological effects on cancer cell growth and BRD4 functions. Among 21 compounds developed here, we identified aminocyclopropenone 1n (ACP-1n) with the strongest inhibitory effects on the growth of the cancer cell line HCT116. ACP-1n blocked BRD4 functions by preventing its phase separation ability both in vitro and in vivo, attenuating the expression levels of BRD4-driven MYC. Notably, ACP-1n significantly reduced the nuclear size with concomitant suppression of the level of the NPC protein nucleoporin NUP210. Furthermore, NUP210 is in a BRD4-dependent manner and silencing of NUP210 was sufficient to decrease nucleus size and cellular growth. In conclusion, our findings highlighted an aminocyclopropenone compound as a novel therapeutic drug blocking BRD4 assembly, thereby preventing BRD4-driven oncogenic functions in cancer cells. This study facilitates the development of the next generation of effective and potent inhibitors of epigenetic bromodomains and extra-terminal (BET) protein family.
Subject(s)
Cell Cycle Proteins , Colorectal Neoplasms , Nuclear Pore Complex Proteins , Transcription Factors , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Proliferation , Colorectal Neoplasms/drug therapy , Humans , Nuclear Pore Complex Proteins/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.
Subject(s)
COVID-19/metabolism , COVID-19/virology , Host Microbial Interactions/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Gene Knockdown Techniques , HeLa Cells , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Models, Biological , Nuclear Envelope/metabolism , Nuclear Envelope/virology , Nuclear Pore Complex Proteins/antagonists & inhibitors , Nuclear Pore Complex Proteins/genetics , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor RelA/metabolism , Viral Nonstructural Proteins/geneticsABSTRACT
SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain (RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2 complex appears as an all-RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction with receptor ACE2 and sEV-based decoy in real time using HS-AFM.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Extracellular Vesicles/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Humans , Hydrogen-Ion Concentration , Lipid Bilayers/metabolism , Microscopy, Atomic Force , Protein Binding , Protein Conformation , Protein Domains , Protein Subunits , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Temperature , Virus InternalizationABSTRACT
To reconstruct systematically hyperactive transcription factor (TF)-dependent transcription networks in squamous cell carcinomas (SCCs), a computational method (ELMER) was applied to 1293 pan-SCC patient samples, and 44 hyperactive SCC TFs were identified. As a top candidate, DLX5 exhibits a notable bifurcate re-configuration of its bivalent promoter in cancer. Specifically, DLX5 maintains a bivalent state in normal tissues; its promoter is hypermethylation, leading to DLX5 transcriptional silencing in esophageal adenocarcinoma (EAC). In stark contrast, DLX5 promoter gains active histone marks and becomes transcriptionally activated in ESCC, which is directly mediated by SOX2. Functionally, silencing of DLX5 substantially inhibits SCC viability both in vitro and in vivo. Mechanistically, DLX5 cooperates with TP63 in regulating â¼2000 enhancers and promoters, which converge on activating cancer-promoting pathways. Together, our data establish a novel and strong SCC-promoting factor and elucidate a new epigenomic mechanism - bifurcate chromatin re-configuration - during cancer development.
Subject(s)
Adenocarcinoma/genetics , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Homeodomain Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Adenocarcinoma/pathology , Animals , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Proliferation/genetics , DNA Methylation/genetics , Esophageal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic/genetics , Heterografts , Humans , Male , Mice , Middle Aged , Promoter Regions, Genetic/geneticsABSTRACT
Biomolecules may undergo liquid-liquid phase separation (LLPS) to spatiotemporally compartmentalize and regulate diverse biological processes. Because the number of tools to directly probe LLPS is limited (ie. FRAP, FRET, fluorescence microscopy, fluorescence anisotropy, circular dichroism, etc.), the physicochemical traits of phase-separated condensates remain largely elusive. Here, we introduce a light-switching dipyrene probe (Pyr-A) that forms monomers in either hydrophobic or viscous environments, and intramolecular excimers in aqueous solutions. By exploiting their distinct fluorescence emission spectra, we used fluorescent microscopic imaging to study phase-separated condensates formed by in vitro protein droplets and membraneless intracellular organelles (centrosomes). Ratiometric measurement of excimer and monomer fluorescence intensities showed that protein droplets became hydrophobic and viscous as their size increased. Moreover, centrosomes became hydrophobic and viscous during maturation. Our results show that Pyr-A is a valuable tool to characterize LLPS and enhance our understanding of phase separation underlying biological functions.
ABSTRACT
Squamous cell carcinomas (SCCs) comprise one of the most common histologic types of human cancer. Transcriptional dysregulation of SCC cells is orchestrated by tumor protein p63 (TP63), a master transcription factor (TF) and a well-researched SCC-specific oncogene. In the present study, both Gene Set Enrichment Analysis (GSEA) of SCC patient samples and in vitro loss-of-function assays establish fatty-acid metabolism as a key pathway downstream of TP63. Further studies identify sterol regulatory element binding transcription factor 1 (SREBF1) as a central mediator linking TP63 with fatty-acid metabolism, which regulates the biosynthesis of fatty-acids, sphingolipids (SL), and glycerophospholipids (GPL), as revealed by liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics. Moreover, a feedback co-regulatory loop consisting of SREBF1/TP63/Kruppel like factor 5 (KLF5) is identified, which promotes overexpression of all three TFs in SCCs. Downstream of SREBF1, a non-canonical, SCC-specific function is elucidated: SREBF1 cooperates with TP63/KLF5 to regulate hundreds of cis-regulatory elements across the SCC epigenome, which converge on activating cancer-promoting pathways. Indeed, SREBF1 is essential for SCC viability and migration, and its overexpression is associated with poor survival in SCC patients. Taken together, these data shed light on mechanisms of transcriptional dysregulation in cancer, identify specific epigenetic regulators of lipid metabolism, and uncover SREBF1 as a potential therapeutic target and prognostic marker in SCC.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Esophageal Neoplasms/metabolism , Head and Neck Neoplasms/metabolism , Kruppel-Like Transcription Factors/metabolism , Lipid Metabolism/genetics , Lung Neoplasms/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Acetylation , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Chromatin Immunoprecipitation Sequencing , Chromatography, Liquid , Epigenomics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Esophageal Neoplasms/genetics , Fatty Acids/biosynthesis , Fatty Acids/metabolism , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/genetics , Histones/metabolism , Humans , Kruppel-Like Transcription Factors/genetics , Lung Neoplasms/genetics , Regulatory Elements, Transcriptional , Signal Transduction/genetics , Sphingolipids/biosynthesis , Sphingolipids/metabolism , Sterol Regulatory Element Binding Protein 1/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tandem Mass Spectrometry , Transcription Factors/genetics , Transcriptome/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
DNA-histone interaction is always perturbed by epigenetic regulators to regulate gene expression. Direct visualization of this interaction is yet to be achieved. By using high-speed atomic force microscopy (HS-AFM), we have observed the dynamic DNA-histone H2A interaction. HS-AFM movies demonstrate the globular core and disordered tail of H2A. DNA-H2A formed the classic "beads-on-string" conformation on poly-l-lysine (PLL) and lipid substrates. Notably, a short-linearized double-stranded DNA (dsDNA), resembling an inchworm, wrapped around a single H2A protein only observed on the lipid substrate. Such a phenomenon does not occur for plasmid DNA or linearized long dsDNA on the same substrate. Strong adsorption of PLL substrate resulted in poor dynamic DNA-H2A interaction. Nonetheless, short-linearized dsDNA-H2A formed stable wrapping with a "diamond ring" topology on the PLL substrate. Reversible liquid-liquid phase separation (LLPS) of the DNA-H2A aggregate was visualized by manipulating salt concentrations. Collectively, our study suggest that HS-AFM is feasible for investigating epigenetically modified DNA-histone interactions.