RESUMEN
Human stem cells and derivatives transplantation are widely used to treat nervous system diseases, while the fate determination of transplanted cells is not well elucidated. To explore cell fate changes of human brain organoids before and after transplantation, human brain organoids are transplanted into prefrontal cortex (PFC) and hippocampus (HIP), respectively. Single-cell sequencing is then performed. According to time-series sample comparison, transplanted cells mainly undergo neural development at 2 months post-transplantation (MPT) and then glial development at 4MPT, respectively. A different brain region sample comparison shows that organoids grafted to PFC have obtained cell fate close to those of host cells in PFC, other than HIP, which may be regulated by the abundant expression of dopamine (DA) and acetylcholine (Ach) in PFC. Meanwhile, morphological complexity of human astrocyte grafts is greater in PFC than in HIP. DA and Ach both activate the calcium activity and increase morphological complexity of astrocytes in vitro. This study demonstrates that human brain organoids receive host niche factor regulation after transplantation, resulting in the alignment of grafted cell fate with implanted brain regions, which may contribute to a better understanding of cell transplantation and regenerative medicine.
Asunto(s)
Organoides , Transcriptoma , Humanos , Organoides/metabolismo , Organoides/citología , Organoides/trasplante , Transcriptoma/genética , Encéfalo/metabolismo , Análisis de la Célula Individual/métodos , Diferenciación Celular/genética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/citología , Hipocampo/metabolismoRESUMEN
The alteration of gene expression is not restricted to transcriptional regulation but includes a variety of post-transcriptional mechanisms, however, the role of the latter underlying many diseases remains relatively unknown. By utilizing an RNA-Seq dataset of 1510 brain samples from individuals with autism spectrum disorder (ASD), bipolar disorder (BD), schizophrenia (SCZ), and controls, we assessed the contribution of post-transcriptional dysregulation and identified top perturbators accountable for transcriptomic changes of expression in neuropsychiatric disorders. Around 30% of the variability in expression can be attributed to post-transcriptional dysregulation. Interestingly, RNA stability tended to decrease in SCZ and BD, leading to the inhibition of neurogenesis and neural differentiation, while the increase in ASD, resulted in enhanced activity of apoptosis. This finding implicated contrasting pathologies involving RNA stability among neuropsychiatric disorders. An RNA binding protein (RBP)-ELAVL3 - is predicted to be significantly involved in the disruption of RNA stability in all three disorders. To validate, we knocked down its expression in cerebral organoids. Not only differentially expressed genes in ELAVL3-knockdown covered a considerable proportion of predicted targets in three disorders, we also found neurogenesis was significantly affected, given the diminished proliferation and consequently the reduced size of the organoids. Our study extends the current understanding of the link between post-transcriptional regulation and neuropsychiatric disorders and provides new therapeutic targets for early intervention.
RESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disease, whose pathogenetic complexity was strongly associated with aging/smoking and poorly understood. METHODS: Here we performed single-cell RNA sequencing (scRNA-seq) analysis of 66,610 cells from COPD and age-stratified control lung tissues of donors with different smoking histories to prioritize cell types most perturbed in COPD lungs in aging/smoking dependent or independent manner. By performing an array of advanced bioinformatic analyses, such as gene set enrichment analysis, trajectory analysis, cell-cell interactions analysis, regulatory potential analysis, weighted correlation network analysis, functional interaction analysis, and gene set variation analysis, we integrated cell-type-level alterations into a system-level malfunction and provided a more clarified COPD pathological model containing specific mechanisms by which aging and smoking facilitate COPD development. Finally, we integrated the publicly available scRNA-seq data of 9 individuals, resulting in a total of 110,931 cells, and replicated the analyses to enhance the credibility of our findings. RESULTS: Our study pointed to enrichment of COPD molecular alteration in monocytes, which further induced a previously unrecognized pro-inflammatory effect on alveolar epithelial cells. In addition, aged monocytes and club cells facilitated COPD development via maintaining an autoimmune airway niche. Unexpectedly, macrophages, whose defect to resolve inflammation was long-recognized in COPD pathogenesis, primarily induced an imbalance of sphingolipids rheostat in a smoking-dependent way. These findings were validated in a meta-analysis including other public single-cell transcriptomic data. CONCLUSIONS: In sum, our study provided a clarified view of COPD pathogenesis and demonstrated the potential of targeting monocytes in COPD diagnosis and treatment.
Asunto(s)
Monocitos , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Anciano , Monocitos/metabolismo , Transcriptoma , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Pulmón/metabolismo , Perfilación de la Expresión GénicaRESUMEN
Meiosis entry during spermatogenesis requires reprogramming from mitotic to meiotic gene expression profiles. Transcriptional regulation has been extensively studied in meiosis entry, but gain of function for master transcription factors is insufficient to down-regulate mitotic genes. RNA helicase YTHDC2 and its partner MEIOC emerge as essential posttranscriptional regulators of meiotic entry. However, it is unclear what governs the RNA binding specificity of YTHDC2/MEIOC. Here, we identified RNA binding protein RBM46 as a component of the YTHDC2/MEIOC complex. Testis-specific Rbm46 knockout in mice causes infertility with defective mitotic-to-meiotic transition, phenocopying global Ythdc2 or Meioc knockout. RBM46 binds to 3' UTR of mitotic transcripts within 100 nucleotides from YTHDC2 U-rich motifs and targets these transcripts for degradation. Dysregulated RBM46 expression is associated with human male fertility disorders. These findings establish the RBM46/YTHDC2/MEIOC complex as the major posttranscriptional regulator responsible for down-regulating mitotic transcripts during meiosis entry in mammalian spermatogenesis, with implications for understanding meiosis-related fertility disorders.
Asunto(s)
Meiosis , Proteínas de Unión al ARN , Espermatogénesis , Animales , Humanos , Masculino , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismo , Meiosis/genética , Ratones Noqueados , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
Genetic variants within or near the transcription factor 4 gene ( TCF4) are robustly implicated in psychiatric disorders including schizophrenia. However, the biological pleiotropy poses considerable obstacles to dissect the potential relationship between TCF4 and those highly heterogeneous diseases. Through integrative transcriptomic analysis, we demonstrated that TCF4 is preferentially expressed in cortical interneurons during early brain development. Therefore, disruptions of interneuron development might be the underlying contribution of TCF4 perturbation to a range of neurodevelopmental disorders. Here, we performed chromatin immunoprecipitation sequencing (ChIP-seq) of TCF4 on human medial ganglionic eminence-like organoids (hMGEOs) to identify genome-wide TCF4 binding sites, followed by integration of multi-omics data from human fetal brain. We observed preferential expression of the isoform TCF4-B over TCF4-A. De novo motif analysis found that the identified 5916 TCF4 binding sites are significantly enriched for the E-box sequence. The predicted TCF4 targets in general have positively correlated expression levels with TCF4 in the cortical interneurons, and are primarily involved in biological processes related to neurogenesis. Interestingly, we found that TCF4 interacts with non-bHLH proteins such as FOS/JUN, which may underlie the functional specificity of TCF4 in hMGEOs. This study highlights the regulatory role of TCF4 in interneuron development and provides compelling evidence to support the biological rationale linking TCF4 to the developing cortical interneuron and psychiatric disorders.
RESUMEN
This study aimed to investigate the effect and mechanism of valproic acid (VPA) on the neurosphere formation in rat embryonic cortical cells. We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells (NSCs). We found a time- and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment. Further RNA-seq analysis demonstrated that the upregulated TGFß1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation. Consistently, the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542, an inhibitor of TGFß1 receptor. Moreover, in a coculture system, NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis. Taken together, our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFß1, which might be a novel therapeutic strategy for neurological disorders.
RESUMEN
Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type-specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Corteza Cerebral/metabolismo , Síndrome de Down/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neurogénesis , Organoides/metabolismo , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular , Síndrome de Down/genética , Humanos , Quinasas p21 Activadas/genéticaRESUMEN
The transition from meiotic spermatocytes to postmeiotic haploid germ cells constitutes an essential step in spermatogenesis. The epigenomic regulatory mechanisms underlying this transition remain unclear. Here, we find a prominent transcriptomic switch from the late spermatocytes to the early round spermatids during the meiotic-to-postmeiotic transition, which is associated with robust histone acetylation changes across the genome. Among histone deacetylases (HDACs) and acetyltransferases, we find that HDAC3 is selectively expressed in the late meiotic and early haploid stages. Three independent mouse lines with the testis-specific knockout of HDAC3 show infertility and defects in meiotic exit with an arrest at the late stage of meiosis or early stage of round spermatids. Stage-specific RNA-seq and histone acetylation ChIP-seq analyses reveal that HDAC3 represses meiotic/spermatogonial genes and activates postmeiotic haploid gene programs during meiotic exit, with associated histone acetylation alterations. Unexpectedly, abolishing HDAC3 catalytic activity by missense mutations in the nuclear receptor corepressor (NCOR or SMRT) does not cause infertility, despite causing histone hyperacetylation as HDAC3 knockout, demonstrating that HDAC3 enzyme activity is not required for spermatogenesis. Motif analysis of the HDAC3 cistrome in the testes identified SOX30, which has a similar spatiotemporal expression pattern as HDAC3 during spermatogenesis. Depletion of SOX30 in the testes abolishes the genomic recruitment of the HDAC3 to the binding sites. Collectively, these results establish the SOX30/HDAC3 signaling as a key regulator of the transcriptional program in a deacetylase-independent manner during the meiotic-to-postmeiotic transition in spermatogenesis.
Asunto(s)
Fertilidad/genética , Regulación de la Expresión Génica , Histona Desacetilasas/fisiología , Meiosis/genética , Espermatogénesis/genética , Activación Transcripcional , Acetilación , Animales , Reprogramación Celular/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción SOX/metabolismo , Espermátides/citología , Espermátides/metabolismo , Testículo/metabolismoRESUMEN
BACKGROUND: Genetic biomarkers of lung cancer (LC) susceptibility may provide a basis for treatment and prevention. This study analyzed an association between SNPs (single nucleotide polymorphisms) in the complementary region of the 3'-UTR (3' untranslated region) of microRNAs of the gene RIPK1 (receptor-interacting serine/threonine-protein kinase 1) and LC among an adult Han Chinese population aged younger than 60 years. Also explored the effect of regulation of the RIPK1 gene via rs17548629 and microRNA-1197 on the occurrence of LC. METHODS: RIPK1 variants (rs17548629, rs77736895) were determined in a population of 571 adults (younger than 60 years) with LC, and 609 gender- and age-matched healthy individuals. Bioinformatics methods predicted the microRNAs bound to rs17548629. Dual luciferase reporter assay was performed to confirm the presence of both rs17548629 and the predicted microRNA. RESULTS: A mutation (T) of rs17548629 was associated with an increased risk for LC in this population under the codominant and recessive genetic models. The risk of lung adenocarcinoma in rs17548629 mutant carriers was 1.769-fold higher than that of the wildtype. In vitro, the luciferase activity of co-transfected mutant psiCHECK2-RIPK1 and microRNA-1197 mimics was less than that of the group transfected with microRNA-1197 mimics only. Factorial analysis indicated interactions between microRNA-1197 mimics and genotypes of rs17548629. CONCLUSION: A mutation (T) of rs17548629 may increase the risk of LC/lung adenocarcinoma in adult Han populations younger than 60 years. When carrying the T allele, rs17548629 may be the target of hsa-miR-1197. This mutation may affect transcriptional level of the RIPK1, thereby promoting the occurrence of LC.
RESUMEN
Idiopathic pulmonary fibrosis (IPF) has been widely accepted as an aging-related fatal lung disease with a therapeutic impasse, largely a consequence of the complex and polygenic gene architecture underlying the molecular pathology of IPF. Here, by conducting an integrative network analysis on the largest IPF case-control RNA-seq dataset to date, we attributed the systems-level alteration in IPF to disruptions in a handful of biological processes including cell migration, transforming growth factor-ß (TGF-ß) signaling and extracellular matrix (ECM), and identified klotho (KL), a typical anti-aging molecule, as a potential master regulator of those disease-relevant processes. Following experiments showed reduced Kl in isolated pulmonary fibroblasts from bleomycin-exposed mice, and demonstrated that recombinant KL effectively mitigated pulmonary fibrosis in an ex vivo model and alleviated TGF-ß-induced pulmonary fibroblasts activation, migration, and ECM production in vitro, which was partially ascribed to FOXF1 and CAV1, two highly co-expressed genes of KL in the IPF. Overall, KL appears to be a vital regulator during pulmonary fibrosis. Given that administration of exogenous KL is a feasible treatment strategy, our work highlighted a promising target gene that could be easily manipulated, leaving the field well placed to further explore the therapeutic potential of KL for IPF.
Asunto(s)
Movimiento Celular/fisiología , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Animales , Proliferación Celular/fisiología , Biología Computacional , Matriz Extracelular/patología , Fibroblastos/patología , Glucuronidasa/genética , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/patología , Proteínas Klotho , Pulmón/patología , Masculino , Ratones , ARN Interferente Pequeño , Transducción de SeñalRESUMEN
BACKGROUND: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells. RESULTS: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites. CONCLUSIONS: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.
Asunto(s)
Células Madre Germinales Adultas/metabolismo , ARN Helicasas/genética , Espermatozoides/metabolismo , Animales , Perfilación de la Expresión Génica , Masculino , Ratones , ARN Helicasas/metabolismoRESUMEN
Glial activation plays a pivotal role in morphine tolerance. This study investigated effects of Mas-related gene (Mrg) C receptor on morphine-induced activation of microglia and astrocytes in the spinal cord and its underlying mechanisms. Intrathecal administration of morphine (20 µg, daily) for 6 days induced a great decline in morphine antinociception and increased expression of glial fibrillary acidic protein and OX-42 in the spinal dorsal horn. These changes were greatly attenuated by the intermittent coinjection of bovine adrenal medulla 8-22 (BAM8-22, 1 nmol), a specific agonist of MrgC receptor. These modulatory effects were accompanied by the reduction of P2X4 and interleukin-1ß expressions in the spinal dorsal horn. Chronic morphine increased the expression of fractalkine in medium- and small-sized neurons of dorsal root ganglia (DRG). Treatment with BAM8-22 inhibited these changes as well as an increase in Toll-like receptor 4 (TLR4) protein in DRG. Chronic treatment of DRG explant cultures with morphine (3.3 µM, 5 days) increased the levels of fractalkine mRNA. Application of BAM8-22 (10 nM) for 60 minutes completely blocked the increase of fractalkine mRNA induced by morphine. Our findings indicate that the inhibition of morphine tolerance by MrgC receptor was associated with the modulation of astrocytes and microglia in the spinal dorsal horn and fractalkine and TLR4 expressions in DRG. As MrgC receptor is exclusively located in DRG, intermittent combination of MrgC receptor agonist could be a promising adjunct with limited side effects for chronic use of opiates.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Ganglios Espinales/metabolismo , Morfina/administración & dosificación , Neuroglía/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Bovinos , Ganglios Espinales/efectos de los fármacos , Inyecciones Espinales , Masculino , Neuroglía/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/administración & dosificación , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistasRESUMEN
Human GABAergic interneurons (GIN) are implicated in normal brain function and in numerous mental disorders. However, the generation of functional human GIN subtypes from human pluripotent stem cells (hPSCs) has not been established. By expressing LHX6, a transcriptional factor that is critical for GIN development, we induced hPSCs to form GINs, including somatostatin (SST, 29%) and parvalbumin (PV, 21%) neurons. Our RNAseq results also confirmed the alteration of GIN identity with the overexpression of LHX6. Five months after transplantation into the mouse brain, the human GABA precursors generated increased population of SST and PV neurons by overexpressing LHX6. Importantly, the grafted human GINs exhibited functional electrophysiological properties and even fast-spiking-like action potentials. Thus, expression of the single transcription factor LHX6 under our GIN differentiation condition is sufficient to robustly induce human PV and SST subtypes.
Asunto(s)
Proteínas con Homeodominio LIM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Parvalbúminas/metabolismo , Somatostatina/metabolismo , Factores de Transcripción/metabolismo , Potenciales de Acción , Animales , Animales Recién Nacidos , Tipificación del Cuerpo , Diferenciación Celular , Línea Celular , Perfilación de la Expresión Génica , Humanos , Interneuronas/citología , Interneuronas/metabolismo , Ratones SCID , Neuronas/citología , Neuronas/trasplante , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Prosencéfalo/citología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Meiosis/fisiología , Factores de Transcripción SOX/metabolismo , Espermátides/metabolismo , Espermatogénesis/fisiología , Testículo/metabolismo , Iniciación de la Transcripción Genética/fisiología , Animales , Perfilación de la Expresión Génica , Masculino , Ratones , Elementos de Respuesta/fisiología , Factores de Transcripción SOX/genética , Espermátides/citología , Testículo/citologíaRESUMEN
BACKGROUND: Monoallelic expression of autosomal genes has been implicated in human psychiatric disorders. However, there is a paucity of allelic expression studies in human brain cells at the single cell and genome wide levels. RESULTS: In this report, we reanalyzed a previously published single-cell RNA-seq dataset from several postmortem human brains and observed pervasive monoallelic expression in individual cells, largely in a random manner. Examining single nucleotide variants with a predicted functional disruption, we found that the "damaged" alleles were overall expressed in fewer brain cells than their counterparts, and at a lower level in cells where their expression was detected. We also identified many brain cell type-specific monoallelically expressed genes. Interestingly, many of these cell type-specific monoallelically expressed genes were enriched for functions important for those brain cell types. In addition, function analysis showed that genes displaying monoallelic expression and correlated expression across neuronal cells from different individual brains were implicated in the regulation of synaptic function. CONCLUSIONS: Our findings suggest that monoallelic gene expression is prevalent in human brain cells, which may play a role in generating cellular identity and neuronal diversity and thus increasing the complexity and diversity of brain cell functions.
Asunto(s)
Alelos , Encéfalo/citología , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/ß-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in ß-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of ß-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, ß-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.
Asunto(s)
Anomalías Cardiovasculares/genética , Síndrome de DiGeorge/genética , Modelos Animales de Enfermedad , Proteínas de Dominio T Box/genética , beta Catenina/genética , Animales , Apoptosis/genética , Anomalías Cardiovasculares/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/genética , Síndrome de DiGeorge/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Dominio T Box/metabolismo , Tronco Arterial/citología , Tronco Arterial/embriología , Tronco Arterial/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Individuals with 22q11.2 Deletion Syndrome (22q11.2 DS) are a specific high-risk group for developing schizophrenia (SZ), schizoaffective disorder (SAD) and autism spectrum disorders (ASD). Several genes in the deleted region have been implicated in the development of SZ, e.g., PRODH and DGCR8. However, the mechanistic connection between these genes and the neuropsychiatric phenotype remains unclear. To elucidate the molecular consequences of 22q11.2 deletion in early neural development, we carried out RNA-seq analysis to investigate gene expression in early differentiating human neurons derived from induced pluripotent stem cells (iPSCs) of 22q11.2 DS SZ and SAD patients. METHODS: Eight cases (ten iPSC-neuron samples in total including duplicate clones) and seven controls (nine in total including duplicate clones) were subjected to RNA sequencing. Using a systems level analysis, differentially expressed genes/gene-modules and pathway of interests were identified. Lastly, we related our findings from in vitro neuronal cultures to brain development by mapping differentially expressed genes to BrainSpan transcriptomes. RESULTS: We observed ~2-fold reduction in expression of almost all genes in the 22q11.2 region in SZ (37 genes reached p-value < 0.05, 36 of which reached a false discovery rate < 0.05). Outside of the deleted region, 745 genes showed significant differences in expression between SZ and control neurons (p < 0.05). Function enrichment and network analysis of the differentially expressed genes uncovered converging evidence on abnormal expression in key functional pathways, such as apoptosis, cell cycle and survival, and MAPK signaling in the SZ and SAD samples. By leveraging transcriptome profiles of normal human brain tissues across human development into adulthood, we showed that the differentially expressed genes converge on a sub-network mediated by CDC45 and the cell cycle, which would be disrupted by the 22q11.2 deletion during embryonic brain development, and another sub-network modulated by PRODH, which could contribute to disruption of brain function during adolescence. CONCLUSIONS: This study has provided evidence for disruption of potential molecular events in SZ patient with 22q11.2 deletion and related our findings from in vitro neuronal cultures to functional perturbations that can occur during brain development in SZ.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Neuronas/patología , Trastornos Psicóticos/genética , Esquizofrenia/genética , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Línea Celular , Redes Reguladoras de Genes , Humanos , Trastornos Psicóticos/patología , Trastornos Psicóticos/fisiopatología , Esquizofrenia/patología , Esquizofrenia/fisiopatologíaRESUMEN
OBJECTIVE: Long noncoding RNAs (lncRNA) represent a growing class of noncoding genes with diverse cellular functions. We previously reported on SENCR, an lncRNA that seems to support the vascular smooth muscle cell (VSMC) contractile phenotype. However, information about the VSMC-specific lncRNAs regulated by myocardin (MYOCD)/serum response factor, the master switch for VSMC differentiation, is unknown. APPROACH AND RESULTS: To define novel lncRNAs with functions related to VSMC differentiation, we performed RNA sequencing in human coronary artery SMCs that overexpress MYOCD. Several novel lncRNAs showed altered expression with MYOCD overexpression and one, named MYOcardin-induced Smooth muscle LncRNA, Inducer of Differentiation (MYOSLID), was activated by MYOCD and selectively expressed in VSMCs. MYOSLID was a direct transcriptional target of both MYOCD/serum response factor and transforming growth factor-ß/SMAD pathways. Functional studies revealed that MYOSLID promotes VSMC differentiation and inhibits VSMC proliferation. MYOSLID showed reduced expression in failed human arteriovenous fistula samples compared with healthy veins. Although MYOSLID did not affect gene expression of transcription factors, such as serum response factor and MYOCD, its depletion in VSMCs disrupted actin stress fiber formation and blocked nuclear translocation of MYOCD-related transcription factor A (MKL1). Finally, loss of MYOSLID abrogated transforming growth factor-ß1-induced SMAD2 phosphorylation. CONCLUSIONS: We have demonstrated that MYOSLID, the first human VSMC-selective and serum response factor/CArG-dependent lncRNA, is a novel modulator in amplifying the VSMC differentiation program, likely through feed-forward actions of both MKL1 and transforming growth factor-ß/SMAD pathways.
Asunto(s)
Diferenciación Celular , Desarrollo de Músculos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/metabolismo , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Derivación Arteriovenosa Quirúrgica , Proliferación Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas Nucleares/genética , Fenotipo , Fosforilación , ARN Largo no Codificante/genética , Factor de Respuesta Sérica/genética , Transducción de Señal , Proteína Smad2/metabolismo , Fibras de Estrés/metabolismo , Factores de Tiempo , Transactivadores/genética , Transcripción Genética , Transfección , Factor de Crecimiento Transformador beta1/metabolismo , VasoconstricciónRESUMEN
PURPOSE: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets. EXPERIMENTAL DESIGN: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line-derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA). RESULTS: Rac GTPase-activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease. CONCLUSIONS: The RACGAP1-STAT3-survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676-86. ©2016 AACR.