ABSTRACT
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. OBJECTIVES: To evaluate the efficacy and safety of intravenous (IV) gentamicin readthrough therapy in patients with RDEB harbouring nonsense mutations. The primary outcomes were increased expression of C7 in patients' skin and safety assessments (ototoxicity, nephrotoxicity, autoimmune response); secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) scoring system. METHODS: An open-label pilot trial to assess two different IV gentamicin regimens between August 2018 and March 2020 with follow-up through to 180â days post-treatment was carried out. Three patients with RDEB with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin 7.5â mg kg-1 daily for 14â days and two of the three patients further received 7.5â mg kg-1 IV gentamicin twice weekly for 12 weeks. Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. RESULTS: After gentamicin treatment, skin biopsies from all three patients (age range 18-28â years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted for at least 6â months post-treatment. At 1 and 3â months post-treatment, 100% of the monitored wounds exhibited > 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of the patients assessed with the EBDASI, all exhibited decreased total activity scores 3â months post-treatment. All three patients completed the study; no adverse effects or anti-C7 antibodies were detected. CONCLUSIONS: IV gentamicin induced the readthrough of nonsense mutations in patients with RDEB and restored functional C7 in their skin, enhanced wound healing and improved clinical parameters. IV gentamicin may be a safe, efficacious, low-cost and readily available treatment for this population of patients with RDEB.
Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and life-threatening inherited skin disease that causes widespread skin blisters that heal with scarring. RDEB affects around 1 in every 100,000 individuals globally. The condition is caused by a mutation in the gene coding for type VII collagen (C7), resulting in a deficiency of C7. C7 is a vital component of the skin as it is responsible for holding the skin's upper two layers together. To date, there are no approved systemic treatments that can cure RDEB. This study, from the United States, aimed to evaluate the effectiveness and safety of intravenous (medicine delivered directly into a patient's vein) gentamicin (an antibiotic) for people with RDEB who have nonsense mutations in their genes (a type of mutation that prevents the production of complete proteins by introducing an inappropriate 'stop signal'). We gave gentamicin to three patients with RDEB every day for 14â days, and two of the three patients further received intravenous gentamicin twice a week for 12 weeks. After gentamicin treatment, all three patients showed increased expression of C7. With both regimens, the new C7 stayed for at least 6â months after the treatment. At 1 and 3â months after treatment, 100% of the wounds being monitored in the patients had closed by more than 85%. All three patients completed the study, and no side-effects were experienced. In conclusion, intravenous gentamicin increased the production of C7 and improved wound healing and quality of life in patients with RDEB carrying nonsense mutations. Intravenous gentamicin may offer a safe, effective, low-cost and readily available therapy in patients with RDEB.
Subject(s)
Codon, Nonsense , Collagen Type VII , Epidermolysis Bullosa Dystrophica , Gentamicins , Humans , Gentamicins/administration & dosage , Gentamicins/adverse effects , Epidermolysis Bullosa Dystrophica/drug therapy , Epidermolysis Bullosa Dystrophica/genetics , Collagen Type VII/genetics , Collagen Type VII/immunology , Pilot Projects , Male , Female , Adult , Adolescent , Treatment Outcome , Young Adult , Wound Healing/drug effects , Skin/pathology , Skin/drug effects , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/adverse effects , Drug Administration ScheduleABSTRACT
BACKGROUND: The ring-shaped cohesin complex is an important factor for the formation of chromatin loops and topologically associating domains (TADs) by loop extrusion. However, the regulation of association between cohesin and chromatin is poorly understood. In this study, we use super-resolution imaging to reveal the unique role of cohesin subunit RAD21 in cohesin loading and chromatin structure regulation. RESULTS: We directly visualize that up-regulation of RAD21 leads to excessive chromatin loop extrusion into a vermicelli-like morphology with RAD21 clustered into foci and excessively loaded cohesin bow-tying a TAD to form a beads-on-a-string-type pattern. In contrast, up-regulation of the other four cohesin subunits results in even distributions. Mechanistically, we identify that the essential role of RAD21 is attributed to the RAD21-loader interaction, which facilitates the cohesin loading process rather than increasing the abundance of cohesin complex upon up-regulation of RAD21. Furthermore, Hi-C and genomic analysis reveal how RAD21 up-regulation affects genome-wide higher-order chromatin structure. Accumulated contacts are shown at TAD corners while inter-TAD interactions increase after vermicelli formation. Importantly, we find that in breast cancer cells, the expression of RAD21 is aberrantly high with poor patient survival and RAD21 forms beads in the nucleus. Up-regulated RAD21 in HeLa cells leads to compartment switching and up-regulation of cancer-related genes. CONCLUSIONS: Our results provide key insights into the molecular mechanism by which RAD21 facilitates the cohesin loading process and provide an explanation to how cohesin and loader work cooperatively to promote chromatin extrusion, which has important implications in construction of three-dimensional genome organization.
Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Humans , HeLa Cells , Cell Cycle Proteins/genetics , Chromatin , DNA-Binding Proteins , CohesinsABSTRACT
As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.
Subject(s)
Chromatin , Heterochromatin , Humans , Chromatin/metabolism , Heterochromatin/metabolism , HeLa Cells , Cell Nucleolus/metabolism , Cell NucleusABSTRACT
Natural killer (NK) cells are lymphocytes that are involved in controlling tumors or microbial infections through the production of interferon gamma (IFN-γ). Granulocyte colony-stimulating factor (G-CSF) inhibits IFN-γ secretion by NK cells, but the mechanism underlying this effect remains unclear. Here, by comparing the multi-omics profiles of human NK cells before and after in vivo G-CSF treatment, we identify a pathway that is activated in response to G-CSF treatment, which suppresses IFN-γ secretion in NK cells. Specifically, glucocorticoid receptors (GRs) activated by G-CSF inhibit secretion of IFN-γ by promoting interactions between SOCS1 promoters and enhancers, as well as increasing the expression of SOCS1. Experiments in mice confirm that G-CSF treatment significantly downregulates IFN-γ secretion and upregulates GR and SOCS1 expression in NK cells. In addition, GR blockade by the antagonist RU486 significantly reverses the effects of G-CSF, demonstrating that GRs upregulate SOCS1 and inhibit the production of IFN-γ by NK cells.
Subject(s)
Interferon-gamma , Killer Cells, Natural , Animals , Gene Expression , Granulocyte Colony-Stimulating Factor/metabolism , Granulocyte Colony-Stimulating Factor/pharmacology , Humans , Interferon-gamma/metabolism , Killer Cells, Natural/metabolism , Mice , Suppressor of Cytokine Signaling 1 Protein/metabolism , Suppressor of Cytokine Signaling Proteins/metabolismABSTRACT
Chromatin is spatially organized into three-dimensional structures at different levels including A/B compartments, topologically associating domains and loops. The canonical CTCF-mediated loop extrusion model can explain the formation of loops. However, the organization mechanisms underlying long-range chromatin interactions such as interactions between A-A compartments are still poorly understood. Here we show that different from the canonical loop extrusion model, RYBP-mediated phase separation of CTCF organizes inter-A compartment interactions. Based on this model, we designed and verified an induced CTCF phase separation system in embryonic stem cells (ESCs), which facilitated inter-A compartment interactions, improved self-renewal of ESCs and inhibited their differentiation toward neural progenitor cells. These findings support a novel and non-canonical role of CTCF in organizing long-range chromatin interactions via phase separation.
Subject(s)
Chromatin , Neural Stem Cells , CCCTC-Binding Factor/metabolism , Cell Differentiation , Chromatin Assembly and Disassembly , Embryonic Stem Cells/metabolism , Neural Stem Cells/metabolismABSTRACT
Graft-versus-host disease (GVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Inflammatory signaling pathways promote T-cell activation and are involved in the pathogenesis of GVHD. Suppressor of cytokine signaling 1 (SOCS1) is a critical negative regulator for several inflammatory cytokines. However, its regulatory role in T-cell activation and GVHD has not been elucidated. Multiomics analysis of the transcriptome and chromatin structure of granulocyte-colony-stimulating-factor (G-CSF)-administered hyporesponsive T cells from healthy donors reveal that G-CSF upregulates SOCS1 by reorganizing the chromatin structure around the SOCS1 locus. Parallel in vitro and in vivo analyses demonstrate that SOCS1 is critical for restraining T cell activation. Loss of Socs1 in T cells exacerbates GVHD pathogenesis and diminishes the protective role of G-CSF in GVHD mouse models. Further analysis shows that SOCS1 inhibits T cell activation not only by inhibiting the colony-stimulating-factor 3 receptor (CSF3R)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway, but also by restraining activation of the inflammasome signaling pathway. Moreover, high expression of SOCS1 in T cells from patients correlates with low acute GVHD occurrence after HSCT. Overall, these findings identify that SOCS1 is critical for inhibiting T cell activation and represents a potential target for the attenuation of GVHD.
Subject(s)
Graft vs Host Disease , Suppressor of Cytokine Signaling 1 Protein , T-Lymphocytes , Animals , Chromatin , Graft vs Host Disease/etiology , Graft vs Host Disease/genetics , Granulocyte Colony-Stimulating Factor/pharmacology , Mice , Suppressor of Cytokine Signaling 1 Protein/genetics , Systems Biology/methods , T-Lymphocytes/metabolism , Transplantation, Homologous/adverse effectsABSTRACT
The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.
Subject(s)
Chromatin , Lamin Type B , Chromosomes , Genome , Humans , Lamin Type B/geneticsABSTRACT
BACKGROUND: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. RESULTS: We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. CONCLUSION: Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.
Subject(s)
Chromatin/chemistry , DNA Replication , G1 Phase Cell Cycle Checkpoints/genetics , Proliferating Cell Nuclear Antigen/genetics , Replication Origin , Transcription, Genetic , CCCTC-Binding Factor/antagonists & inhibitors , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Cell Line, Tumor , Chromatin Assembly and Disassembly , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression , HeLa Cells , Humans , In Situ Hybridization, Fluorescence , Optical Imaging , Osteoblasts/cytology , Osteoblasts/metabolism , Proliferating Cell Nuclear Antigen/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/metabolismABSTRACT
Topological-associated domains (TADs) are thought to be relatively stable across cell types, although some TAD reorganization has been observed during cellular differentiation. However, little is known about the mechanisms through which TAD reorganization affects cell fate or how master transcription factors affect TAD structures during cell fate transitions. Here, we show extensive TAD reorganization during somatic cell reprogramming, which is correlated with gene transcription and changes in cellular identity. Manipulating TAD reorganization promotes reprogramming, and the dynamics of concentrated chromatin loops in OCT4 phase separated condensates contribute to TAD reorganization. Disrupting OCT4 phase separation attenuates TAD reorganization and reprogramming, which can be rescued by fusing an intrinsically disordered region (IDR) to OCT4. We developed an approach termed TAD reorganization-based multiomics analysis (TADMAN), which identified reprogramming regulators. Together, these findings elucidate a role and mechanism of TAD reorganization, regulated by OCT4 phase separation, in cellular reprogramming.
Subject(s)
Cellular Reprogramming , Chromatin , Octamer Transcription Factor-3/metabolism , Cell DifferentiationABSTRACT
Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.
Subject(s)
Long Interspersed Nucleotide Elements , Repetitive Sequences, Nucleic Acid , Animals , Cluster Analysis , Long Interspersed Nucleotide Elements/genetics , Mice , RNA , Repetitive Sequences, Nucleic Acid/genetics , RetroelementsABSTRACT
Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.
Subject(s)
Brain/embryology , Evolution, Molecular , Fetus/embryology , Genome , Organogenesis/genetics , Animals , Base Sequence , Chromatin/metabolism , DNA Transposable Elements/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental , Humans , Macaca mulatta , Mice , Species Specificity , Synteny/genetics , Transcription Factors/metabolismABSTRACT
Gene expression in response to stimuli underlies many fundamental processes. However, how transcription is regulated under these scenarios is largely unknown. Here, we find a previously unknown role of nuclear actin in transcriptional regulation. The RNA-seq data reveal that nuclear actin is required for the serum-induced transcriptional program. Using super-resolution imaging, we found a remarkable enhancement of RNA polymerase II (Pol II) clustering upon serum stimulation, and this enhancement requires nuclear actin. Pol II clusters colocalized with the serum-response genes and nuclear actin filaments upon serum stimulation. Furthermore, N-WASP is required for serum-enhanced Pol II clustering. N-WASP phase-separated with Pol II and nuclear actin. In addition to serum stimulation, nuclear actin also enhanced Pol II clustering upon interferon-γ treatment. Together, our work unveils that nuclear actin promotes the formation of transcription factory on inducible genes, acting as a general mechanism underlying the rapid response to environmental cues.
Subject(s)
Actins , RNA Polymerase II , Actins/metabolism , Cell Nucleus/metabolism , Cluster Analysis , RNA Polymerase II/metabolism , Transcription, GeneticABSTRACT
Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients' skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients' blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Cell Adhesion Molecules/metabolism , Codon, Nonsense/genetics , Epidermolysis Bullosa, Junctional/drug therapy , Epidermolysis Bullosa, Junctional/genetics , Gentamicins/administration & dosage , Signal Transduction/drug effects , Wound Healing/drug effects , Administration, Cutaneous , Anti-Bacterial Agents/adverse effects , Cell Survival/drug effects , Cells, Cultured , Child , Child, Preschool , Epidermolysis Bullosa, Junctional/pathology , Female , Follow-Up Studies , Gentamicins/adverse effects , Humans , Infant , Keratinocytes/metabolism , Male , Skin/metabolism , Treatment Outcome , KalininABSTRACT
As a special kind of delicate light-controllable genetically encoded optical device, reversibly photoswitchable fluorescent proteins (RSFPs) have been widely applied in many fields, especially various kinds of advanced nanoscopy approaches in recent years. However, there are still necessities for exploring novel RSFPs with specific biochemical or photophysical properties not only for bioimaging or biosensing applications but also for fluorescent protein (FP) mechanisms study and further knowledge-based molecular sensors or optical actuators' rational design and evolution. Besides previously reported GMars-Q and GMars-T variants, herein, we reported the development and applications of other RSFPs from GMars family, especially some featured RSFPs with desired optical properties. In the current work, in vitro FP purification, spectra measurements, and live-cell RESOLFT nanoscopy approaches were applied to characterize the basic properties and test the imaging performances of the selected RSFPs. As demonstrated, GMars variants such as GMars-A, GMars-G, or remarkable photofatigue-resistant GMars-L were found with beneficial properties to be capable of parallelized RESOLFT nanoscopy in living cells, while other featured GMars variants such as dark GMars-P may be a good candidate for further biosensor or actuator design and applications.
Subject(s)
Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Cell Line, Tumor , Escherichia coli/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/radiation effects , Humans , Light , Microscopy, Fluorescence/methodsABSTRACT
The BiFC (bimolecular fluorescence complementation) assay and BiFC combined with FRET (fluorescence resonance energy transfer) technique have become important tools for molecular interaction studies in live cells. However, the real detection and cellular imaging performances of most existing red fluorescent protein-derived BiFC assays still suffer from relatively low ensemble brightness, high cytotoxicity, the red fluorescent proteins being prone-to-aggregation or severe residual dimerization, inefficient complementation and slow maturation at 37 °C physiological temperature in live mammalian cells. We developed a BiFC assay based on a recently evolved truly monomeric red fluorescent protein (FP) mScarlet-I with excellent cellular performances such as low cytotoxicity, fast and efficient chromophore maturation and the highest in-cell brightness among all previously reported monomeric red fluorescent proteins. In this work, a classic ß-Fos/ß-Jun constitutive heterodimerization model and a rapamycin-inducible FRB/FKBP interaction system were used to establish and test the performance of the mScarlet-I-based BiFC assay in live mammalian cells. Furthermore, simply by adopting the large-Stokes-shift fluorescent protein mAmetrine as the donor, ß-Jun-ß-Fos-NFAT1 ternary protein complex formation could be readily and efficiently detected and visualized with minimal spectral cross-talk in live HeLa cells by combining live-cell sensitized-emission FRET measurement with the mScarlet-I-based BiFC assay. The currently established BiFC assay in this work was also shown to be able to detect and visualize various protein-protein interactions (PPIs) at different subcellular compartments with high specificity and sensitivity at 37 °C physiological temperature in live mammalian cells.
Subject(s)
Luminescent Proteins/chemistry , NFATC Transcription Factors/metabolism , Protein Multimerization , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Fluorescence , Fluorescence Resonance Energy Transfer/methods , HeLa Cells , Humans , Microscopy, Fluorescence/methods , Red Fluorescent ProteinABSTRACT
Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and â¼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.
Subject(s)
Cell Adhesion Molecules , Codon, Nonsense , Epidermolysis Bullosa, Junctional , Gentamicins/pharmacology , Keratinocytes/metabolism , Mutagenesis/drug effects , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Epidermolysis Bullosa, Junctional/genetics , Epidermolysis Bullosa, Junctional/metabolism , Epidermolysis Bullosa, Junctional/pathology , HEK293 Cells , Humans , Integrin alpha6beta4/genetics , Integrin alpha6beta4/metabolism , Keratinocytes/pathology , KalininABSTRACT
The chromosomes in eukaryotic cells are highly folded and organized to form dynamic three-dimensional (3D) structures. In recent years, many technologies including chromosome conformation capture (3C) and 3C-based technologies (Hi-C, ChIA-PET) have been developed to investigate the 3D structure of chromosomes. These technologies are enabling research on how gene regulatory events are affected by the 3D genome structure, which is increasingly implicated in the regulation of gene expression and cellular functions. Importantly, many diseases are associated with genetic variations, most of which are located in non-coding regions. However, it is difficult to determine the mechanisms by which these variations lead to diseases. With 3D genome technologies, we can now better determine the consequences of non-coding genome alterations via their impact on chromatin interactions and structures in cancer and other diseases. In this review, we introduce the various 3D genome technologies, with a focus on their application to cancer and disease research, as well as future developments to extend their utility.
Subject(s)
Chromosome Structures/genetics , Chromosome Structures/physiology , Genomic Instability/genetics , Chromatin/genetics , Chromatin/physiology , Chromosomes/genetics , Computational Biology/methods , DNA/metabolism , DNA Breaks, Double-Stranded , Disease/genetics , Genome/physiology , Genomic Instability/physiology , Humans , Imaging, Three-Dimensional/methods , Sequence Analysis, DNA/methods , SyndromeABSTRACT
The CRISPR/Cas9 system has made significant contributions to genome editing, gene regulation and chromatin studies in recent years. High-throughput and systematic investigations into the multiplexed biological systems require simultaneous expression and coordinated functioning of multiple sgRNAs. However, current cotransfection based sgRNA coexpression systems remain inefficient, and virus-based transfection approaches are relatively costly and labor intensive. Here we established a vector-independent method allowing multiple sgRNA expression cassettes to be assembled in series into a single plasmid. This synthetic biology-based strategy excels in its efficiency, controllability and scalability. Taking the flexibility advantage of this all-in-one sgRNA expressing system, we further explored its applications in single nonrepetitive genomic locus imaging as well as coordinated gene regulation in live cells. With its full potency, our method will facilitate the research in understanding genome structure, function and dynamics.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , RNA, Guide, Kinetoplastida/metabolism , HEK293 Cells , Humans , In Situ Hybridization, Fluorescence , Microscopy, Fluorescence , Mucin-4/genetics , Plasmids/genetics , Plasmids/metabolism , Promoter Regions, Genetic , RNA, Guide, Kinetoplastida/genetics , SOXB1 Transcription Factors/geneticsABSTRACT
As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.
Subject(s)
Luminescent Proteins/metabolism , Protein Interaction Mapping/methods , Animals , COS Cells , Chlorocebus aethiops , Fluorescence , Fluorescence Resonance Energy Transfer , HeLa Cells , Humans , Microscopy, Fluorescence/methods , Protein BindingABSTRACT
BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS: A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS: Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin. CONCLUSION: Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION: ClinicalTrials.gov NCT02698735. FUNDING: Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.