RESUMEN
Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.
Asunto(s)
Apoptosis/genética , Carcinoma Ductal Pancreático/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Expresión Génica , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Mutaciones Letales Sintéticas , Carcinoma Ductal Pancreático/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
After kidney transplantation (KT), donor-specific hyporesponsiveness (DSH) of recipient T cells develops over time. Recently, apoptosis was identified as a possible underlying mechanism. In this study, both transcriptomic profiles and complete V(D)J variable regions of TR transcripts from individual alloreactive T cells of kidney transplant recipients were determined with single-cell RNA sequencing. Alloreactive T cells were identified by CD137 expression after stimulation of peripheral blood mononuclear cells obtained from KT recipients (N = 7) prior to and 3-5 years after transplantation with cells of their donor or a third party control. The alloreactive T cells were sorted, sequenced and the transcriptome and T cell receptor profiles were analyzed using unsupervised clustering. Alloreactive T cells retain a highly polyclonal T Cell Receptor Alpha/Beta repertoire over time. Post transplantation, donor-reactive CD4+ T cells had a specific downregulation of genes involved in T cell cytokine-mediated pathways and apoptosis. The CD8+ donor-reactive T cell profile did not change significantly over time. Single-cell expression profiling shows that activated and pro-apoptotic donor-reactive CD4+ T cell clones are preferentially lost after transplantation in stable kidney transplant recipients.
Asunto(s)
Trasplante de Riñón , Trasplante de Riñón/efectos adversos , Leucocitos Mononucleares , Receptores de Antígenos de Linfocitos T , Apoptosis , Análisis de Secuencia de ARNRESUMEN
The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.
Asunto(s)
Cromatina/ultraestructura , Elementos de Facilitación Genéticos/genética , Enfermedad de Hirschsprung/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Facies , Regulación de la Expresión Génica/genética , Enfermedad de Hirschsprung/patología , Proteínas de Homeodominio/genética , Humanos , Discapacidad Intelectual/patología , Ratones , Microcefalia/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/ultraestructura , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.
Asunto(s)
Anomalías Múltiples/enzimología , Anomalías Múltiples/genética , Colon/anomalías , Genes Recesivos , Seudoobstrucción Intestinal/enzimología , Seudoobstrucción Intestinal/genética , Mutación/genética , Quinasa de Cadena Ligera de Miosina/genética , Vejiga Urinaria/anomalías , Secuencia de Bases , Colon/enzimología , Femenino , Homocigoto , Humanos , Masculino , Linaje , Vejiga Urinaria/enzimologíaRESUMEN
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.
Asunto(s)
Anomalías Múltiples/genética , Autoantígenos/fisiología , Colon/anomalías , Proteínas del Citoesqueleto/fisiología , Seudoobstrucción Intestinal/genética , Proteínas Musculares/fisiología , Vejiga Urinaria/anomalías , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Codón sin Sentido , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Recién Nacido , Ratones , Contracción Muscular/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso/fisiologíaRESUMEN
BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Proteínas Hedgehog/genética , Enfermedad de Hirschsprung/genética , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteína Gli3 con Dedos de Zinc/genética , Animales , Células COS , Chlorocebus aethiops , Familia , Femenino , Predisposición Genética a la Enfermedad , Variación Genética , Células HEK293 , Humanos , Masculino , Morfolinos , Países Bajos , Linaje , Isoformas de Proteínas , Proto-Oncogenes Mas , Análisis de Secuencia de ADN , Transducción de Señal , Pez CebraRESUMEN
Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin γ-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis.
Asunto(s)
Anomalías Múltiples/genética , Actinas/genética , Colon/anomalías , Mucosa Intestinal/metabolismo , Seudoobstrucción Intestinal/genética , Contracción Muscular/genética , Músculo Liso/metabolismo , Mutación Missense , Vejiga Urinaria/anomalías , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Actinas/química , Actinas/metabolismo , Colon/metabolismo , Colon/patología , Resultado Fatal , Femenino , Expresión Génica , Heterocigoto , Humanos , Recién Nacido , Seudoobstrucción Intestinal/metabolismo , Seudoobstrucción Intestinal/patología , Intestinos/patología , Masculino , Simulación de Dinámica Molecular , Músculo Liso/patología , Linaje , Multimerización de Proteína , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Adulto JovenRESUMEN
PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA in maternal blood is highly sensitive for detecting fetal trisomies 21, 18, and 13. Using a genome-wide approach, other chromosome anomalies can also be detected. We report on the origin, frequency, and clinical significance of these other chromosome aberrations found in pregnancies at risk for trisomy 21, 18, or 13.MethodsWhole-genome shallow massively parallel sequencing was used and all autosomes were analyzed.ResultsIn 78 of 2,527 cases (3.1%) NIPS was indicative of trisomy 21, 18, or 13, and in 41 (1.6%) of other chromosome aberrations. The latter were of fetal (n = 10), placental (n = 22), maternal (n = 1) or unknown (n = 7). One case lacked cytogenetic follow-up. Nine of the 10 fetal cases were associated with an abnormal phenotype. Thirteen of the 22 (59%) placental aberrations were associated with fetal congenital anomalies and/or poor fetal growth (Asunto(s)
Aberraciones Cromosómicas
, Trastornos de los Cromosomas/diagnóstico
, Trastornos de los Cromosomas/genética
, Pruebas Genéticas
, Diagnóstico Prenatal
, Trisomía
, Variaciones en el Número de Copia de ADN
, Femenino
, Pruebas Genéticas/métodos
, Genómica/métodos
, Humanos
, Placenta/metabolismo
, Embarazo
, Resultado del Embarazo
, Diagnóstico Prenatal/métodos
, Secuenciación Completa del Genoma
RESUMEN
BACKGROUND: The genetic bases of PD in sub-Saharan African (SSA) populations remain poorly characterized, and analysis of SSA families with PD might lead to the discovery of novel disease-related genes. OBJECTIVES: To investigate the clinical features and identify the disease-causing gene in a black South African family with 3 members affected by juvenile-onset parkinsonism and intellectual disability. METHODS: Clinical evaluation, neuroimaging studies, whole-exome sequencing, homozygosity mapping, two-point linkage analysis, and Sanger sequencing of candidate variants. RESULT: A homozygous 28-nucleotide frameshift deletion in the PTRHD1 coding region was identified in the 3 affected family members and linked to the disease with genome-wide significant evidence. PTRHD1 was recently nominated as the disease-causing gene in two Iranian families, each containing 2 siblings with similar phenotypes and homozygous missense mutations. CONCLUSION: Together with the previous reports, we provide conclusive evidence that loss-of-function mutations in PTRHD1 cause autosomal-recessive juvenile parkinsonism and intellectual disability. © 2018 International Parkinson and Movement Disorder Society.
Asunto(s)
Salud de la Familia , Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Mutación/genética , Trastornos Parkinsonianos/genética , Adulto , África del Sur del Sahara , Análisis Mutacional de ADN , Femenino , Humanos , Discapacidad Intelectual/complicaciones , Masculino , Trastornos Parkinsonianos/complicacionesRESUMEN
Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.
Asunto(s)
Plaquetas/citología , Megacariocitos/citología , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Animales , Azepinas/química , Plaquetas/metabolismo , Médula Ósea/metabolismo , Citometría de Flujo , Lectinas Tipo C/metabolismo , Ratones , Ratones Noqueados , Naftalenos/química , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteoma , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Bazo/metabolismo , Trombocitopenia/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Mitosis , Familia de Multigenes , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transcriptoma , CohesinasRESUMEN
BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.
Asunto(s)
Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Proteínas Supresoras de Tumor/genética , Adolescente , Niño , Sitios Genéticos/genética , Genómica , Humanos , Persona de Mediana Edad , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma , Animales , Humanos , RatonesRESUMEN
Finding genes for complex diseases has been the goal of many genetic studies. Most of these studies have been successful by searching for genes and mutations in rare familial cases, by screening candidate genes and by performing genome wide association studies. However, only a small fraction of the total genetic risk for these complex genetic diseases can be explained by the identified mutations and associated genetic loci. In this review we focus on Hirschsprung disease (HSCR) as an example of a complex genetic disorder. We describe the genes identified in this congenital malformation and postulate that both common 'low penetrant' variants in combination with rare or private 'high penetrant' variants determine the risk on HSCR, and likely, on other complex diseases. We also discuss how new technological advances can be used to gain further insights in the genetic background of complex diseases. Finally, we outline a few steps to develop functional assays in order to determine the involvement of these variants in disease development.
Asunto(s)
Variación Genética , Enfermedad de Hirschsprung/genética , Modelos Biológicos , Animales , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedad de Hirschsprung/patología , HumanosRESUMEN
Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.
Asunto(s)
Biomarcadores de Tumor/genética , Inmunohistoquímica , Melanoma/genética , Mutación , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Neoplasias de la Úvea/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Estimación de Kaplan-Meier , Masculino , Melanoma/mortalidad , Melanoma/patología , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Úvea/mortalidad , Neoplasias de la Úvea/patologíaRESUMEN
Memory T cells provide long-lasting defense responses through their ability to rapidly reactivate, but how they efficiently "recall" an inflammatory transcriptional program remains unclear. Here, we show that human CD4+ memory T helper 2 (TH2) cells carry a chromatin landscape synergistically reprogrammed at both one-dimensional (1D) and 3D levels to accommodate recall responses, which is absent in naive T cells. In memory TH2 cells, recall genes were epigenetically primed through the maintenance of transcription-permissive chromatin at distal (super)enhancers organized in long-range 3D chromatin hubs. Precise transcriptional control of key recall genes occurred inside dedicated topologically associating domains ("memory TADs"), in which activation-associated promoter-enhancer interactions were preformed and exploited by AP-1 transcription factors to promote rapid transcriptional induction. Resting memory TH2 cells from patients with asthma showed premature activation of primed recall circuits, linking aberrant transcriptional control of recall responses to chronic inflammation. Together, our results implicate stable multiscale reprogramming of chromatin organization as a key mechanism underlying immunological memory and dysfunction in T cells.
Asunto(s)
Cromatina , Regulación de la Expresión Génica , Humanos , Cromatina/genética , Factores de Transcripción/genética , Regiones Promotoras GenéticasRESUMEN
Functional perturbation and action mechanism studies have shown that the transcription factor Zeb2 controls cell fate decisions, differentiation, and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs), Zeb2's mRNA/protein upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce Flag-V5 epitope-tagged Zeb2 protein from one endogenous allele. Using chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we mapped 2432 DNA-binding sites for this tagged Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major binding site maps promoter-proximal to Zeb2 itself. The homozygous deletion of this site demonstrates that autoregulation of Zeb2 is necessary to elicit the appropriate Zeb2-dependent effects in ESC-to-NPC differentiation. We have also cross-referenced all the mapped Zeb2 binding sites with previously obtained transcriptome data from Zeb2 perturbations in ESC-derived NPCs, GABAergic interneurons from the ventral forebrain of mouse embryos, and stem/progenitor cells from the post-natal ventricular-subventricular zone (V-SVZ) in mouse forebrain, respectively. Despite the different characteristics of each of these neurogenic systems, we found interesting target gene overlaps. In addition, our study also contributes to explaining developmental disorders, including Mowat-Wilson syndrome caused by ZEB2 deficiency, and also other monogenic syndromes.
Asunto(s)
Neuronas , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Animales , Ratones , Sitios de Unión , ADN/química , ADN/metabolismo , Homocigoto , Neuronas/metabolismo , Neuronas/patología , Eliminación de Secuencia , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Humanos , Transcriptoma/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodosRESUMEN
BACKGROUND: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants' gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. METHODS: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. RESULTS: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p < 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. CONCLUSIONS: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. TRIAL REGISTRATION: Netherlands Trial Registry Identifier: NL7815 19/06/2019.