RESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is linked to abnormal derepression of the transcription activator DUX4. This effect is localized to a low percentage of cells, requiring single-cell analysis. However, single-cell/nucleus RNA-seq cannot fully capture the transcriptome of multinucleated large myotubes. To circumvent these issues, we use multiplexed error-robust fluorescent in situ hybridization (MERFISH) spatial transcriptomics that allows profiling of RNA transcripts at a subcellular resolution. We simultaneously examined spatial distributions of 140 genes, including 24 direct DUX4 targets, in in vitro differentiated myotubes and unfused mononuclear cells (MNCs) of control, isogenic D4Z4 contraction mutant and FSHD patient samples, as well as the individual nuclei within them. We find myocyte nuclei segregate into two clusters defined by the expression of DUX4 target genes, which is exclusively found in patient/mutant nuclei, whereas MNCs cluster based on developmental states. Patient/mutant myotubes are found in "FSHD-hi" and "FSHD-lo" states with the former signified by high DUX4 target expression and decreased muscle gene expression. Pseudotime analyses reveal a clear bifurcation of myoblast differentiation into control and FSHD-hi myotube branches, with variable numbers of DUX4 target-expressing nuclei found in multinucleated FSHD-hi myotubes. Gene coexpression modules related to extracellular matrix and stress gene ontologies are significantly altered in patient/mutant myotubes compared with the control. We also identify distinct subpathways within the DUX4 gene network that may differentially contribute to the disease transcriptomic phenotype. Taken together, our MERFISH-based study provides effective gene network profiling of multinucleated cells and identifies FSHD-induced transcriptomic alterations during myoblast differentiation.
Assuntos
Fibras Musculares Esqueléticas , Distrofia Muscular Facioescapuloumeral , Mioblastos , Análise de Célula Única , Transcriptoma , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Humanos , Mioblastos/metabolismo , Análise de Célula Única/métodos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Diferenciação Celular/genética , Hibridização in Situ Fluorescente , Perfilação da Expressão Gênica/métodosRESUMO
Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.
RESUMO
Single-cell RNA-seq (scRNA-seq) has revolutionized modern genomics, but the large size of myotubes and myofibers has restricted use of scRNA-seq in skeletal muscle. For the study of muscle, single-nucleus RNA-seq (snRNA-seq) has emerged not only as an alternative to scRNA-seq, but as a novel method providing valuable insights into multinucleated cells such as myofibers. Nuclei within myofibers specialize at junctions with other cell types such as motor neurons. Nuclear heterogeneity plays important roles in certain diseases such as muscular dystrophies. We survey current methods of high-throughput single cell and subcellular resolution transcriptomics, including single-cell and single-nucleus RNA-seq and spatial transcriptomics, applied to satellite cells, myoblasts, myotubes and myofibers. We summarize the major myonuclei subtypes identified in homeostatic and regenerating tissue including those specific to fiber type or at junctions with other cell types. Disease-specific nucleus populations were found in two muscular dystrophies, FSHD and Duchenne muscular dystrophy, demonstrating the importance of performing transcriptome studies at the single nucleus level in muscle.
RESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is the second most common genetic myopathy, characterized by slowly progressing and highly heterogeneous muscle wasting with a typical onset in the late teens/early adulthood [1]. Although the etiology of the disease for both FSHD type 1 and type 2 has been attributed to gain-of-toxic function stemming from aberrant DUX4 expression, the exact pathogenic mechanisms involved in muscle wasting have yet to be elucidated [2-4]. The 2021 FSHD International Research Congress, held virtually on June 24-25, convened over 350 researchers and clinicians to share the most recent advances in the understanding of the disease mechanism, discuss the proliferation of interventional strategies and refinement of clinical outcome measures, including results from the ReDUX4 trial, a phase 2b clinical trial of losmapimod in FSHD [NCT04003974].
Assuntos
Distrofia Muscular Facioescapuloumeral , Adolescente , Adulto , Proteínas de Homeodomínio/genética , Humanos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Distrofia Muscular Facioescapuloumeral/metabolismoRESUMO
Facioscapulohumeral dystrophy (FSHD) is associated with the upregulation of the DUX4 transcription factor and its target genes. However, low-frequency DUX4 upregulation in patient myocytes is difficult to detect and examining the relationship and dynamics of DUX4 and target gene expression has been challenging. Using RNAScope in situ hybridization with highly specific probes, we detect the endogenous DUX4 and target gene transcripts in situ in patient skeletal myotubes during 13-day differentiation in vitro. We found that the endogenous DUX4 transcripts primarily localize as foci in one or two nuclei as compared with the accumulation of the recombinant DUX4 transcripts in the cytoplasm. We also found the continuous increase of DUX4 and target gene-positive myotubes after Day 3, arguing against its expected immediate cytotoxicity. Interestingly, DUX4 and target gene expression become discordant later in differentiation with the increase of DUX4-positive/target gene-negative as well as DUX4-negative/target gene-positive myotubes. Depletion of DUX4-activated transcription factors, DUXA and LEUTX, specifically repressed a DUX4-target gene, KDM4E, later in differentiation, suggesting that after the initial activation by DUX4, target genes themselves contribute to the maintenance of downstream gene expression. Together, the study provides important new insights into the dynamics of the DUX4 transcriptional network in FSHD patient myocytes.
Assuntos
Distrofia Muscular Facioescapuloumeral , Núcleo Celular/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapuloumeral/genéticaRESUMO
FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.
Assuntos
Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral , RNA-Seq/métodos , Análise de Célula Única/métodos , Estudos de Casos e Controles , Diferenciação Celular , Núcleo Celular/química , Núcleo Celular/classificação , Núcleo Celular/patologia , Células Cultivadas , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequenciamento do ExomaRESUMO
Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase.
Assuntos
Quebras de DNA/efeitos da radiação , Reparo do DNA , DNA/biossíntese , Fase G1/genética , Mitose/genética , Animais , Linhagem Celular , DNA/genética , DNA/efeitos da radiação , Fase G1/efeitos da radiação , Humanos , Raios Infravermelhos/efeitos adversos , Lasers/efeitos adversos , Mitose/efeitos da radiação , PotoroidaeRESUMO
As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Fibras Musculares Esqueléticas , Miócitos Cardíacos , Sarcômeros , Algoritmos , Animais , Células Cultivadas , Humanos , Microscopia de Fluorescência , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Miofibrilas/fisiologia , Ratos , Ratos Sprague-Dawley , Sarcômeros/química , Sarcômeros/ultraestruturaRESUMO
DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.
Assuntos
Dano ao DNA , Reparo do DNA , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Fibroblastos , Glicólise/fisiologia , Células HeLa , Humanos , Células MCF-7 , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Fosforilação Oxidativa , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de SinaisRESUMO
TRF2 (TERF2) binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I), and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by TERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination repair, but not homologous recombination between sister chromatids or non-homologous end-joining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.
Assuntos
Cromátides/metabolismo , Dano ao DNA , Reparo de DNA por Recombinação , Proteína 2 de Ligação a Repetições Teloméricas/genética , Linhagem Celular Tumoral , Cromátides/genética , Ativação Enzimática , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Telomerase/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.
Assuntos
Dano ao DNA , Lasers , Animais , Reparo do DNA , HumanosRESUMO
BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder frequently associated with heterozygous loss-of-function mutations of Nipped-B-like (NIPBL), the human homolog of Drosophila Nipped-B. NIPBL loads cohesin onto chromatin. Cohesin mediates sister chromatid cohesion important for mitosis but is also increasingly recognized as a regulator of gene expression. In CdLS patient cells and animal models, expression changes of multiple genes with little or no sister chromatid cohesion defect suggests that disruption of gene regulation underlies this disorder. However, the effect of NIPBL haploinsufficiency on cohesin binding, and how this relates to the clinical presentation of CdLS, has not been fully investigated. Nipbl haploinsufficiency causes CdLS-like phenotype in mice. We examined genome-wide cohesin binding and its relationship to gene expression using mouse embryonic fibroblasts (MEFs) from Nipbl+/- mice that recapitulate the CdLS phenotype. RESULTS: We found a global decrease in cohesin binding, including at CCCTC-binding factor (CTCF) binding sites and repeat regions. Cohesin-bound genes were found to be enriched for histone H3 lysine 4 trimethylation (H3K4me3) at their promoters; were disproportionately downregulated in Nipbl mutant MEFs; and displayed evidence of reduced promoter-enhancer interaction. The results suggest that gene activation is the primary cohesin function sensitive to Nipbl reduction. Over 50% of significantly dysregulated transcripts in mutant MEFs come from cohesin target genes, including genes involved in adipogenesis that have been implicated in contributing to the CdLS phenotype. CONCLUSIONS: Decreased cohesin binding at the gene regions is directly linked to disease-specific expression changes. Taken together, our Nipbl haploinsufficiency model allows us to analyze the dosage effect of cohesin loading on CdLS development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Perfilação da Expressão Gênica/métodos , Haploinsuficiência , Proteínas/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Metilação de DNA , Síndrome de Cornélia de Lange/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Ativação Transcricional , CoesinasRESUMO
In addition to their mitotic and transcriptional functions, cohesin plays critical roles in DNA damage response (DDR) and repair. Specifically, cohesin promotes homologous recombination (HR) repair of DNA double-strand breaks (DSBs), which is conserved from yeast to humans, and is a critical effector of ATM/ATR DDR kinase-mediated checkpoint control in mammalian cells. Optical laser microirradiation has been instrumental in revealing the damage site-specific functions of cohesin and, more recently, uncovering the unique role of cohesin-SA2, one of the two cohesin complexes uniquely present in higher eukaryotes, in DNA repair in human cells. In this review, we briefly describe what we know about cohesin function and regulation in response to DNA damage, and discuss the optimized laser microirradiation conditions used to analyze cohesin responses to DNA damage in vivo.
Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Biologia Molecular/métodos , Proteínas Nucleares/genética , Animais , Ciclo Celular/efeitos da radiação , Cromátides/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Humanos , Lasers , Reparo de DNA por Recombinação/genética , Reparo de DNA por Recombinação/efeitos da radiação , Saccharomyces cerevisiae/genética , CoesinasRESUMO
Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation.
Assuntos
Diferenciação Celular/genética , Mioblastos/citologia , Análise de Sequência de RNA/métodos , Linhagem Celular , Núcleo Celular/genética , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , Fibras Musculares Esqueléticas/citologia , Mioblastos/fisiologia , Fator Regulador Miogênico 5/genética , RNA Longo não Codificante , Análise de Célula Única/métodosRESUMO
Laser microirradiation is a powerful tool for real-time single-cell analysis of the DNA damage response (DDR). It is often found, however, that factor recruitment or modification profiles vary depending on the laser system employed. This is likely due to an incomplete understanding of how laser conditions/dosages affect the amounts and types of damage and the DDR. We compared different irradiation conditions using a femtosecond near-infrared laser and found distinct damage site recruitment thresholds for 53BP1 and TRF2 correlating with the dose-dependent increase of strand breaks and damage complexity. Low input-power microirradiation that induces relatively simple strand breaks led to robust recruitment of 53BP1 but not TRF2. In contrast, increased strand breaks with complex damage including crosslinking and base damage generated by high input-power microirradiation resulted in TRF2 recruitment to damage sites with no 53BP1 clustering. We found that poly(ADP-ribose) polymerase (PARP) activation distinguishes between the two damage states and that PARP activation is essential for rapid TRF2 recruitment while suppressing 53BP1 accumulation at damage sites. Thus, our results reveal that careful titration of laser irradiation conditions allows induction of varying amounts and complexities of DNA damage that are gauged by differential PARP activation regulating protein assembly at the damage site.
Assuntos
Dano ao DNA , Lasers , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Adulto , Animais , California , Proteínas de Ciclo Celular/metabolismo , Criança , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/metabolismo , Síndrome de Cornélia de Lange/patologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Camundongos , Fenótipo , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , CoesinasRESUMO
Low level light therapy (LLLT) has numerous therapeutic benefits, including improving wound healing, but the precise mechanisms involved are not well established; in particular, the underlying role of cytochrome C oxidase (C-ox) as the primary photoacceptor and the associated biochemical mechanisms still require further investigation. We previously showed the nitric oxide (NO) donating drug nitrosyl-cobinamide (NO-Cbi) enhances wound healing through a cGMP/cGMP-dependent protein kinase/ERK1/2 mechanism. Here, we show that the combination of LLLT and NO-Cbi markedly improves wound healing compared to either treatment alone. LLLT-enhanced wound healing proceeded through an electron transport chain-C-ox-dependent mechanism with a reduction of reactive oxygen species and increased adenosine triphosphate production. C-ox was validated as the primary photoacceptor by three observations: increased oxygen consumption, reduced wound healing in the presence of sodium azide, and disassociation of cyanide, a known C-ox ligand, following LLLT. We conclude that LLLT and NO-Cbi accelerate wound healing through two independent mechanisms, the electron transport chain-C-ox pathway and cGMP signaling, respectively, with both resulting in ERK1/2 activation.
Assuntos
Cobamidas/química , Terapia com Luz de Baixa Intensidade , Cicatrização , Trifosfato de Adenosina/química , Linhagem Celular Tumoral , Proliferação de Células , Cianetos/química , GMP Cíclico/química , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Temperatura Alta , Humanos , Luz , Óxido Nítrico/química , Nitrogênio/química , Consumo de Oxigênio , Fotoquímica , Espécies Reativas de Oxigênio , Transdução de Sinais , Azida Sódica/químicaRESUMO
Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.
Assuntos
Núcleo Celular/genética , Cromatina/genética , Reparo de DNA por Recombinação , Cromatina/química , Quebras de DNA de Cadeia Dupla , Células HeLa , HumanosRESUMO
Although selective binding of 53BP1 to dimethylated histone H4 lysine 20 (H4K20me2) at DNA double-strand breaks (DSBs) is a necessary and pivotal determinant of nonhomologous end joining (NHEJ)-directed repair, the enzymes that generate H4K20me2 at DSBs were unclear. Here, we determined that the PR-Set7 monomethyltransferase (H4K20me1) regulates de novo H4K20 methylation at DSBs. Rapid recruitment of PR-Set7 to DSBs was dependent on the NHEJ Ku70 protein and necessary for NHEJ-directed repair. PR-Set7 monomethyltransferase activity was required, but insufficient, for H4K20me2 and 53BP1 nucleation at DSBs. We determined that PR-Set7-mediated H4K20me1 facilitates Suv4-20 methyltransferase recruitment and catalysis to generate H4K20me2 necessary for 53BP1 binding. The orchestrated and concerted activities of PR-Set7 and Suv4-20 were required for proficient 53BP1 nucleation and DSB repair. This report identifies PR-Set7 as an essential component of NHEJ and implicates PR-Set7 as a central determinant of NHEJ-directed repair early in mammalian DSB repair pathway choice.
Assuntos
Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transporte Ativo do Núcleo Celular , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligação Proteica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as "TIPs") during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends.