Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 61
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Genome Res ; 34(5): 665-679, 2024 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-38777608

RESUMEN

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.


Asunto(s)
Fibras Musculares Esqueléticas , Distrofia Muscular Facioescapulohumeral , Mioblastos , Análisis de la Célula Individual , Transcriptoma , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patología , Distrofia Muscular Facioescapulohumeral/metabolismo , Humanos , Mioblastos/metabolismo , Análisis de la Célula Individual/métodos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Diferenciación Celular/genética , Hibridación Fluorescente in Situ , Perfilación de la Expresión Génica/métodos
2.
Cell ; 132(3): 422-33, 2008 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-18237772

RESUMEN

Cohesins mediate sister chromatid cohesion, which is essential for chromosome segregation and postreplicative DNA repair. In addition, cohesins appear to regulate gene expression and enhancer-promoter interactions. These noncanonical functions remained unexplained because knowledge of cohesin-binding sites and functional interactors in metazoans was lacking. We show that the distribution of cohesins on mammalian chromosome arms is not driven by transcriptional activity, in contrast to S. cerevisiae. Instead, mammalian cohesins occupy a subset of DNase I hypersensitive sites, many of which contain sequence motifs resembling the consensus for CTCF, a DNA-binding protein with enhancer blocking function and boundary-element activity. We find cohesins at most CTCF sites and show that CTCF is required for cohesin localization to these sites. Recruitment by CTCF suggests a rationale for noncanonical cohesin functions and, because CTCF binding is sensitive to DNA methylation, allows cohesin positioning to integrate DNA sequence and epigenetic state.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Factor de Unión a CCCTC , Diferenciación Celular , Línea Celular , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Citocinas/genética , Desoxirribonucleasa I/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Linfocitos T/citología , Linfocitos T/metabolismo , Cohesinas
3.
PLoS Genet ; 16(5): e1008754, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32365093

RESUMEN

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.


Asunto(s)
Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapulohumeral , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Estudios de Casos y Controles , Diferenciación Celular , Núcleo Celular/química , Núcleo Celular/clasificación , Núcleo Celular/patología , Células Cultivadas , Regulación de la Expresión Génica , Células HEK293 , Humanos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos/metabolismo , Mioblastos/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuenciación del Exoma
4.
Hum Mutat ; 42(4): 421-433, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33502067

RESUMEN

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.


Asunto(s)
Distrofia Muscular Facioescapulohumeral , Núcleo Celular/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética
5.
PLoS Comput Biol ; 16(3): e1007676, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32130207

RESUMEN

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.


Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Fibras Musculares Esqueléticas , Miocitos Cardíacos , Sarcómeros , Algoritmos , Animales , Células Cultivadas , Humanos , Microscopía Fluorescente , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/ultraestructura , Miofibrillas/fisiología , Ratas , Ratas Sprague-Dawley , Sarcómeros/química , Sarcómeros/ultraestructura
6.
J Cell Sci ; 131(23)2018 12 05.
Artículo en Inglés | MEDLINE | ID: mdl-30404833

RESUMEN

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.


Asunto(s)
Cromátides/metabolismo , Daño del ADN , Reparación del ADN por Recombinación , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Línea Celular Tumoral , Cromátides/genética , Activación Enzimática , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Telomerasa/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo
7.
Genes Dev ; 26(13): 1473-85, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-22751501

RESUMEN

DNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister chromatid homologous recombination (SCR). Here we show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent small ubiquitin-like modifier (SUMO) accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the nonsumoylatable Scc1 mutant (15KR) maintain sister chromatid cohesion during mitosis but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation.


Asunto(s)
Proteínas Portadoras/metabolismo , Cromátides , Ligasas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Recombinación Genética , Sumoilación , Acetilación , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular , ADN/genética , ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN , Humanos , Ligasas/genética , Mitosis , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética
8.
Nucleic Acids Res ; 44(3): e27, 2016 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-26424850

RESUMEN

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.


Asunto(s)
Daño del ADN , Rayos Láser , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión al Supresor Tumoral P53
9.
Nucleic Acids Res ; 44(21): e158, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27566152

RESUMEN

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.


Asunto(s)
Diferenciación Celular/genética , Mioblastos/citología , Análisis de Secuencia de ARN/métodos , Línea Celular , Núcleo Celular/genética , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , MicroARNs/genética , Fibras Musculares Esqueléticas/citología , Mioblastos/fisiología , Factor 5 Regulador Miogénico/genética , ARN Largo no Codificante , Análisis de la Célula Individual/métodos
10.
Genes Dev ; 24(22): 2531-42, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20966048

RESUMEN

Although the PR-Set7/Set8/KMT5a histone H4 Lys 20 monomethyltransferase (H4K20me1) plays an essential role in mammalian cell cycle progression, especially during G2/M, it remained unknown how PR-Set7 itself was regulated. In this study, we discovered the mechanisms that govern the dynamic regulation of PR-Set7 during mitosis, and that perturbation of these pathways results in defective mitotic progression. First, we found that PR-Set7 is phosphorylated at Ser 29 (S29) specifically by the cyclin-dependent kinase 1 (cdk1)/cyclinB complex, primarily from prophase through early anaphase, subsequent to global accumulation of H4K20me1. While S29 phosphorylation did not affect PR-Set7 methyltransferase activity, this event resulted in the removal of PR-Set7 from mitotic chromosomes. S29 phosphorylation also functions to stabilize PR-Set7 by directly inhibiting its interaction with the anaphase-promoting complex (APC), an E3 ubiquitin ligase. The dephosphorylation of S29 during late mitosis by the Cdc14 phosphatases was required for APC(cdh1)-mediated ubiquitination of PR-Set7 and subsequent proteolysis. This event is important for proper mitotic progression, as constitutive phosphorylation of PR-Set7 resulted in a substantial delay between metaphase and anaphase. Collectively, we elucidated the molecular mechanisms that control PR-Set7 protein levels during mitosis, and demonstrated that its orchestrated regulation is important for normal mitotic progression.


Asunto(s)
Células/citología , Células/enzimología , Regulación Enzimológica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Mitosis/fisiología , Secuencia de Aminoácidos , Proteína Quinasa CDC2/metabolismo , Cromosomas/metabolismo , Células HEK293 , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas , Alineación de Secuencia , Ubiquitinación
11.
Genes Dev ; 23(20): 2400-4, 2009 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-19793863

RESUMEN

HepA-related protein (HARP) (also known as SMARCAL1) is an ATP-driven annealing helicase that catalyzes the formation of dsDNA from complementary Replication protein A (RPA)-bound ssDNA. Here we find that HARP contains a conserved N-terminal motif that is necessary and sufficient for binding to RPA. This RPA-binding motif is not required for annealing helicase activity, but is essential for the recruitment of HARP to sites of laser-induced DNA damage. These findings suggest that the interaction of HARP with RPA increases the concentration of annealing helicase activity in the vicinity of ssDNA regions to facilitate processes such as DNA repair.


Asunto(s)
ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Proteína de Replicación A/metabolismo , Secuencias de Aminoácidos , Animales , Daño del ADN , ADN Helicasas/química , Células HeLa , Humanos , Ratones , Unión Proteica , Estabilidad Proteica
12.
J Biol Chem ; 289(33): 22771-22784, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-24982423

RESUMEN

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.


Asunto(s)
Anafase , Cromosomas de los Mamíferos/metabolismo , Daño del ADN , Células Epiteliales/metabolismo , Riñón/metabolismo , Rayos Láser/efectos adversos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Células Epiteliales/citología , Exodesoxirribonucleasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Histonas/metabolismo , Riñón/citología , Fosforilación , Potoroidae , Proteínas Quinasas/metabolismo
13.
Biochim Biophys Acta ; 1839(3): 191-202, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24269489

RESUMEN

Cohesins are conserved and essential Structural Maintenance of Chromosomes (SMC) protein-containing complexes that physically interact with chromatin and modulate higher-order chromatin organization. Cohesins mediate sister chromatid cohesion and cellular long-distance chromatin interactions affecting genome maintenance and gene expression. Discoveries of mutations in cohesin's subunits and its regulator proteins in human developmental disorders, so-called "cohesinopathies," reveal crucial roles for cohesins in development and cellular growth and differentiation. In this review, we discuss the latest findings concerning cohesin's functions in higher-order chromatin architecture organization and gene regulation and new insight gained from studies of cohesinopathies. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Trastornos de los Cromosomas/metabolismo , Regulación de la Expresión Génica , Animales , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Trastornos de los Cromosomas/genética , Humanos , Cohesinas
14.
Am J Med Genet A ; 167(6): 1179-92, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25899772

RESUMEN

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.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Síndrome de Cornelia de Lange/genética , Regulación del Desarrollo de la Expresión Génica , Mutación , Adulto , Animales , California , Proteínas de Ciclo Celular/metabolismo , Niño , Proteínas Cromosómicas no Histona/metabolismo , Síndrome de Cornelia de Lange/metabolismo , Síndrome de Cornelia de Lange/patología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Ratones , Fenotipo , Transducción de Señal , Pez Cebra/genética , Pez Cebra/metabolismo , Cohesinas
15.
Biophys J ; 107(1): 55-65, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24988341

RESUMEN

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.


Asunto(s)
Núcleo Celular/genética , Cromatina/genética , Reparación del ADN por Recombinación , Cromatina/química , Roturas del ADN de Doble Cadena , Células HeLa , Humanos
16.
Hum Mutat ; 35(8): 998-1010, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24838473

RESUMEN

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri-methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non-4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD.


Asunto(s)
ADN Satélite , Epigénesis Genética , Heterocromatina/metabolismo , Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Mutación , Animales , Secuencia de Bases , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 4 , Cricetinae , Expresión Génica , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Distrofia Muscular Facioescapulohumeral/metabolismo , Distrofia Muscular Facioescapulohumeral/patología , Mioblastos/metabolismo , Mioblastos/patología , Sistemas de Lectura Abierta , Cultivo Primario de Células , Homología de Secuencia de Ácido Nucleico
17.
Am J Med Genet A ; 164A(6): 1384-93, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24504889

RESUMEN

Cornelia de Lange syndrome (CdLS) is the prototype for the cohesinopathy disorders that have mutations in genes associated with the cohesin subunit in all cells. Roberts syndrome is the next most common cohesinopathy. In addition to the developmental implications of cohesin biology, there is much translational and basic research, with progress towards potential treatment for these conditions. Clinically, there are many issues in CdLS faced by the individual, parents and caretakers, professionals, and schools. The following abstracts are presentations from the 5th Cornelia de Lange Syndrome Scientific and Educational Symposium on June 20-21, 2012, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting, Lincolnshire, IL. The research committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts and subsequently disseminates the information to the families. In addition to the basic science and clinical discussions, there were educationally-focused talks related to practical aspects of management at home and in school. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.


Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Anomalías Craneofaciales/genética , Síndrome de Cornelia de Lange/genética , Ectromelia/genética , Hipertelorismo/genética , Proteínas/genética , Acetiltransferasas/genética , Envejecimiento Prematuro/genética , Animales , Cromatina/genética , Trastornos del Conocimiento/genética , Drosophila , Conducta Alimentaria , Haploinsuficiencia , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Humanos , Ratones , Modelos Animales , Proteínas del Grupo Polycomb/genética , Biosíntesis de Proteínas/genética , Homeostasis del Telómero , Pez Cebra , Cohesinas
18.
iScience ; 27(4): 109357, 2024 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-38510139

RESUMEN

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.

19.
J Biol Chem ; 286(20): 17870-8, 2011 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-21454523

RESUMEN

The ß-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica/fisiología , Elementos Aisladores/fisiología , Globinas beta/biosíntesis , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Humanos , Células K562 , Ratones , Mutación , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Globinas beta/genética , Cohesinas
20.
J Cell Biol ; 177(4): 587-97, 2007 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-17502424

RESUMEN

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis.


Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Centrómero/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Acetiltransferasas/fisiología , Células HeLa , Humanos , Acetiltransferasa E N-Terminal , Cohesinas
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA