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1.
Immunity ; 56(10): 2325-2341.e15, 2023 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-37652021

RESUMEN

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.


Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Humanos , Macrófagos/metabolismo , Aterosclerosis/metabolismo , Placa Aterosclerótica/metabolismo , Quimiocinas/metabolismo , Inflamación/metabolismo , Necrosis/metabolismo
2.
Genome Res ; 2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-39255977

RESUMEN

Pleiotropy, measured as expression breadth across tissues, is one of the best predictors for protein sequence and expression conservation. In this study, we investigated its effect on the evolution of cis-regulatory elements (CREs). To this end, we carefully reanalyzed the Epigenomics Roadmap data for nine fetal tissues, assigning a measure of pleiotropic degree to nearly half a million CREs. To assess the functional conservation of CREs, we generated ATAC-seq and RNA-seq data from humans and macaques. We found that more pleiotropic CREs exhibit greater conservation in accessibility, and the mRNA expression levels of the associated genes are more conserved. This trend of higher conservation for higher degrees of pleiotropy persists when analyzing the transcription factor binding repertoire. In contrast, simple DNA sequence conservation of orthologous sites between species tends to be even lower for pleiotropic CREs than for species-specific CREs. Combining various lines of evidence, we propose that the lack of sequence conservation in functionally conserved pleiotropic CREs is owing to within-element compensatory evolution. In summary, our findings suggest that pleiotropy is also a good predictor for the functional conservation of CREs, even though this is not reflected in the sequence conservation of pleiotropic CREs.

3.
Mol Cell ; 65(4): 631-643.e4, 2017 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-28212749

RESUMEN

Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols.


Asunto(s)
Células Madre Embrionarias/química , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Secuencia de Bases , Línea Celular , Simulación por Computador , Análisis Costo-Beneficio , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Ratones , Modelos Económicos , ARN/aislamiento & purificación , Análisis de Secuencia de ARN/economía , Análisis de la Célula Individual/economía
4.
Proc Natl Acad Sci U S A ; 119(30): e2200512119, 2022 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-35857872

RESUMEN

Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.


Asunto(s)
Linfocitos B , Infecciones por Virus de Epstein-Barr , Antígenos Nucleares del Virus de Epstein-Barr , Regulación de la Expresión Génica , Herpesvirus Humano 4 , Proteínas Proto-Oncogénicas c-myc , Transactivadores , Proteínas Virales , Linfocitos B/inmunología , Linfocitos B/virología , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Fase S , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo
5.
Proc Natl Acad Sci U S A ; 119(47): e2206291119, 2022 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-36375074

RESUMEN

Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a subcompatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically up-regulated in infected compared to noninfected nodules. As hydrophobic modification of cell walls is pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA Reductase genes that were specifically activated in the root and/or in the nodule endodermis. Mutant lines in a Fatty acyl-CoA Reductase gene expressed exclusively in the nodule endodermis had decreased deposition of polyesters on this cell layer and increased nodule permeability compared to wild-type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogenase activity, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules.


Asunto(s)
Lotus , Rhizobium , Lotus/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Oxígeno/metabolismo , Poliésteres , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rhizobium/genética , Fijación del Nitrógeno/genética , Simbiosis/genética , Nitrogenasa/metabolismo , Lípidos
6.
J Biol Chem ; 299(4): 103041, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36803961

RESUMEN

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.


Asunto(s)
Proteínas Serina-Treonina Quinasas , Empalme del ARN , Empalme Alternativo , Factores de Empalme de ARN/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Factor de Empalme U2AF/química , Factores de Transcripción/metabolismo , Transición Epitelial-Mesenquimal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo
7.
Cell ; 137(5): 961-71, 2009 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-19490899

RESUMEN

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.


Asunto(s)
Sustitución de Aminoácidos , Ganglios Basales/metabolismo , Evolución Biológica , Factores de Transcripción Forkhead/metabolismo , Vocalización Animal , Animales , Dendritas/metabolismo , Dopamina/metabolismo , Expresión Génica , Heterocigoto , Humanos , Lenguaje , Depresión Sináptica a Largo Plazo , Ratones , Vías Nerviosas , Plasticidad Neuronal , Habla
8.
Nature ; 554(7690): 62-68, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29364867

RESUMEN

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.


Asunto(s)
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Evolución Molecular , Dosificación de Gen , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Alelos , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Progresión de la Enfermedad , Femenino , Genes myc , Genes p53 , Humanos , Masculino , Ratones , Mutación , Subunidad p52 de NF-kappa B/genética , Metástasis de la Neoplasia/genética , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Factores de Transcripción/genética , Transcriptoma/genética , Factor de Crecimiento Transformador beta1/genética , Proteínas Señalizadoras YAP
9.
Nucleic Acids Res ; 50(15): 8491-8511, 2022 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-35904814

RESUMEN

DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Retrovirus Endógenos , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/metabolismo , Animales , Citosina/metabolismo , Desmetilación del ADN , Metilación de ADN , Proteínas de Unión al ADN/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Expresión Génica , Mamíferos/genética , Ratones , Proteínas Proto-Oncogénicas/genética
10.
Hum Mol Genet ; 28(5): 701-717, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30357341

RESUMEN

Genetic disruptions of the forkhead box transcription factor FOXP2 in humans cause an autosomal-dominant speech and language disorder. While FOXP2 expression pattern are highly conserved, its role in specific brain areas for mammalian social behaviors remains largely unknown. Here we studied mice carrying a homozygous cortical Foxp2 deletion. The postnatal development and gross morphological architecture of mutant mice was indistinguishable from wildtype (WT) littermates. Unbiased behavioral profiling of adult mice revealed abnormalities in approach behavior towards conspecifics as well as in the reciprocal responses of WT interaction partners. Furthermore mutant mice showed alterations in acoustical parameters of ultrasonic vocalizations, which also differed in function of the social context. Cell type-specific gene expression profiling of cortical pyramidal neurons revealed aberrant regulation of genes involved in social behavior. In particular Foxp2 mutants showed the downregulation of Mint2 (Apba2), a gene involved in approach behavior in mice and autism spectrum disorder in humans. Taken together these data demonstrate that cortical Foxp2 is required for normal social behaviors in mice.


Asunto(s)
Conducta Animal , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Factores de Transcripción Forkhead/deficiencia , Eliminación de Gen , Proteínas Represoras/deficiencia , Conducta Social , Animales , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/patología , Homocigoto , Ratones , Ratones Noqueados , Neuronas/metabolismo
11.
J Cell Sci ; 131(10)2018 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-29724912

RESUMEN

Developmental processes, such as angiogenesis, are associated with a constant remodeling of the actin cytoskeleton in response to different mechanical stimuli. The mechanosensitive transcription factors MRTF-A (MKL1) and YAP (also known as YAP1) are important mediators of this challenging adaptation process. However, it is as yet unknown whether both pathways respond in an identical or in a divergent manner to a given microenvironmental guidance cue. Here, we use a micropatterning approach to dissect single aspects of cellular behavior in a spatiotemporally controllable setting. Using the exemplary process of angiogenesis, we show that cell-cell contacts and adhesive surface area are shared regulatory parameters of MRTF and YAP on rigid 2D surfaces. By analyzing MRTF and YAP under laminar flow conditions and during cell migration on dumbbell-shaped microstructures, we demonstrate that they exhibit different translocation kinetics. In conclusion, our work promotes the application of micropatterning techniques as a cell biological tool to study mechanosensitive signaling in the context of angiogenesis.


Asunto(s)
Actinas/metabolismo , Vasos Sanguíneos/metabolismo , Técnicas Citológicas/métodos , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mecanotransducción Celular , Actinas/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Vasos Sanguíneos/química , Vasos Sanguíneos/crecimiento & desarrollo , Humanos , Cinética , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Proteínas Señalizadoras YAP
12.
Acta Neuropathol ; 140(2): 121-142, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32562018

RESUMEN

Expansion of a (G4C2)n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Interferones/biosíntesis , Degeneración Nerviosa/patología , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/patología , Animales , Expansión de las Repeticiones de ADN/genética , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/inmunología , Neuronas/patología
13.
Proc Natl Acad Sci U S A ; 114(45): E9579-E9588, 2017 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-29078328

RESUMEN

To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4 Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis.


Asunto(s)
Diferenciación Celular/fisiología , Factor de Transcripción GATA2/metabolismo , Factor de Transcripción GATA3/metabolismo , Placenta/metabolismo , Células Madre Pluripotentes/metabolismo , Factor de Transcripción AP-2/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Línea Celular , Desarrollo Embrionario/fisiología , Células Madre Embrionarias/metabolismo , Femenino , Humanos , Macaca mulatta , Embarazo , Transcriptoma/fisiología , Trofoblastos/metabolismo
14.
J Nat Prod ; 82(7): 1961-1970, 2019 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-31260301

RESUMEN

Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-ß4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.


Asunto(s)
Actinas/metabolismo , Macrólidos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Células Endoteliales de la Vena Umbilical Humana , Humanos , Estructura Molecular , Unión Proteica
15.
Cell Mol Life Sci ; 75(24): 4539-4555, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30206640

RESUMEN

Actin has emerged as a versatile regulator of gene transcription. Cytoplasmatic actin regulates mechanosensitive-signaling pathways such as MRTF-SRF and Hippo-YAP/TAZ. In the nucleus, both polymerized and monomeric actin directly interfere with transcription-associated molecular machineries. Natural actin-binding compounds are frequently used tools to study actin-related processes in cell biology. However, their influence on transcriptional regulation and intranuclear actin polymerization is poorly understood to date. Here, we analyze the effects of two representative actin-binding compounds, Miuraenamide A (polymerizing properties) and Latrunculin B (depolymerizing properties), on transcriptional regulation in primary cells. We find that actin stabilizing and destabilizing compounds inversely shift nuclear actin levels without a direct influence on polymerization state and intranuclear aspects of transcriptional regulation. Furthermore, we identify Miuraenamide A as a potent inducer of G-actin-dependent SRF target gene expression. In contrast, the F-actin-regulated Hippo-YAP/TAZ axis remains largely unaffected by compound-induced actin aggregation. This is due to the inability of AMOTp130 to bind to the amorphous actin aggregates resulting from treatment with miuraenamide. We conclude that actin-binding compounds predominantly regulate transcription via their influence on cytoplasmatic G-actin levels, while transcriptional processes relying on intranuclear actin polymerization or functional F-actin networks are not targeted by these compounds at tolerable doses.


Asunto(s)
Actinas/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Depsipéptidos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Tiazolidinas/farmacología , Transcripción Genética/efectos de los fármacos , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Células 3T3 NIH , Transactivadores/metabolismo
16.
Proc Natl Acad Sci U S A ; 113(19): 5358-63, 2016 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-27118840

RESUMEN

Whereas all mammals have one glutamate dehydrogenase gene (GLUD1), humans and apes carry an additional gene (GLUD2), which encodes an enzyme with distinct biochemical properties. We inserted a bacterial artificial chromosome containing the human GLUD2 gene into mice and analyzed the resulting changes in the transcriptome and metabolome during postnatal brain development. Effects were most pronounced early postnatally, and predominantly genes involved in neuronal development were affected. Remarkably, the effects in the transgenic mice partially parallel the transcriptome and metabolome differences seen between humans and macaques analyzed. Notably, the introduction of GLUD2 did not affect glutamate levels in mice, consistent with observations in the primates. Instead, the metabolic effects of GLUD2 center on the tricarboxylic acid cycle, suggesting that GLUD2 affects carbon flux during early brain development, possibly supporting lipid biosynthesis.


Asunto(s)
Encéfalo/metabolismo , Ciclo del Ácido Cítrico/fisiología , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/metabolismo , Metaboloma/fisiología , Transcriptoma/fisiología , Animales , Glutamato Deshidrogenasa/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad de la Especie
17.
Bioinformatics ; 33(21): 3486-3488, 2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-29036287

RESUMEN

SUMMARY: Power analysis is essential to optimize the design of RNA-seq experiments and to assess and compare the power to detect differentially expressed genes in RNA-seq data. PowsimR is a flexible tool to simulate and evaluate differential expression from bulk and especially single-cell RNA-seq data making it suitable for a priori and posterior power analyses. AVAILABILITY AND IMPLEMENTATION: The R package and associated tutorial are freely available at https://github.com/bvieth/powsimR. CONTACT: vieth@bio.lmu.de or hellmann@bio.lmu.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Análisis de la Célula Individual
18.
Glia ; 65(3): 474-488, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28032919

RESUMEN

A key enzyme in brain glutamate homeostasis is glutamate dehydrogenase (GDH) which links carbohydrate and amino acid metabolism mediating glutamate degradation to CO2 and expanding tricarboxylic acid (TCA) cycle capacity with intermediates, i.e. anaplerosis. Humans express two GDH isoforms, GDH1 and 2, whereas most other mammals express only GDH1. hGDH1 is widely expressed in human brain while hGDH2 is confined to astrocytes. The two isoforms display different enzymatic properties and the nature of these supports that hGDH2 expression in astrocytes potentially increases glutamate oxidation and supports the TCA cycle during energy-demanding processes such as high intensity glutamatergic signaling. However, little is known about how expression of hGDH2 affects the handling of glutamate and TCA cycle metabolism in astrocytes. Therefore, we cultured astrocytes from cerebral cortical tissue of hGDH2-expressing transgenic mice. We measured glutamate uptake and metabolism using [3 H]glutamate, while the effect on metabolic pathways of glutamate and glucose was evaluated by use of 13 C and 14 C substrates and analysis by mass spectrometry and determination of radioactively labeled metabolites including CO2 , respectively. We conclude that hGDH2 expression increases capacity for uptake and oxidative metabolism of glutamate, particularly during increased workload and aglycemia. Additionally, hGDH2 expression increased utilization of branched-chain amino acids (BCAA) during aglycemia and caused a general decrease in oxidative glucose metabolism. We speculate, that expression of hGDH2 allows astrocytes to spare glucose and utilize BCAAs during substrate shortages. These findings support the proposed role of hGDH2 in astrocytes as an important fail-safe during situations of intense glutamatergic activity. GLIA 2017;65:474-488.


Asunto(s)
Astrocitos/metabolismo , Ciclo del Ácido Cítrico/fisiología , Regulación Enzimológica de la Expresión Génica , Glucosa/deficiencia , Glutamato Deshidrogenasa/metabolismo , Ácido Glutámico/metabolismo , Animales , Astrocitos/efectos de los fármacos , Dióxido de Carbono/farmacocinética , Isótopos de Carbono/farmacocinética , Células Cultivadas , Corteza Cerebral/citología , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Deshidrogenasa/genética , Ácido Glutámico/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Tritio/farmacocinética
19.
Proc Natl Acad Sci U S A ; 111(39): 14253-8, 2014 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-25225386

RESUMEN

The acquisition of language and speech is uniquely human, but how genetic changes might have adapted the nervous system to this capacity is not well understood. Two human-specific amino acid substitutions in the transcription factor forkhead box P2 (FOXP2) are outstanding mechanistic candidates, as they could have been positively selected during human evolution and as FOXP2 is the sole gene to date firmly linked to speech and language development. When these two substitutions are introduced into the endogenous Foxp2 gene of mice (Foxp2(hum)), cortico-basal ganglia circuits are specifically affected. Here we demonstrate marked effects of this humanization of Foxp2 on learning and striatal neuroplasticity. Foxp2(hum/hum) mice learn stimulus-response associations faster than their WT littermates in situations in which declarative (i.e., place-based) and procedural (i.e., response-based) forms of learning could compete during transitions toward proceduralization of action sequences. Striatal districts known to be differently related to these two modes of learning are affected differently in the Foxp2(hum/hum) mice, as judged by measures of dopamine levels, gene expression patterns, and synaptic plasticity, including an NMDA receptor-dependent form of long-term depression. These findings raise the possibility that the humanized Foxp2 phenotype reflects a different tuning of corticostriatal systems involved in declarative and procedural learning, a capacity potentially contributing to adapting the human brain for speech and language acquisition.


Asunto(s)
Factores de Transcripción Forkhead/fisiología , Aprendizaje/fisiología , Sustitución de Aminoácidos , Animales , Cuerpo Estriado/fisiología , Dopamina/metabolismo , Femenino , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Humanos , Depresión Sináptica a Largo Plazo , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Destreza Motora/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Especificidad de la Especie , Transcriptoma
20.
Development ; 139(18): 3332-42, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22874921

RESUMEN

MicroRNAs (miRNAs) are rapidly emerging as a new layer of regulation of mammalian brain development. However, most of the miRNA target genes remain unidentified. Here, we explore gene expression profiling upon miRNA depletion and in vivo target validation as a strategy to identify novel miRNA targets in embryonic mouse neocortex. By this means, we find that Foxp2, a transcription factor associated with speech and language development and evolution, is a novel miRNA target. In particular, we find that miR-9 and miR-132 are able to repress ectopic expression of Foxp2 protein by targeting its 3' untranslated region (3'UTR) in vivo. Interestingly, ectopic expression of Foxp2 in cortical projection neurons (a scenario that mimics the absence of miRNA-mediated silencing of Foxp2 expression) delays neurite outgrowth in vitro and impairs their radial migration in embryonic mouse neocortex in vivo. Our results uncover a new layer of control of Foxp2 expression that may be required for proper neuronal maturation.


Asunto(s)
Regiones no Traducidas 3'/genética , Movimiento Celular/fisiología , Factores de Transcripción Forkhead/metabolismo , MicroARNs/metabolismo , Neocórtex/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Animales , Movimiento Celular/genética , Células Cultivadas , Factores de Transcripción Forkhead/genética , Hibridación in Situ , Ratones , MicroARNs/genética , Proteínas Represoras/genética
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