RESUMO
Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.
Assuntos
Neoplasias Hematológicas , Neoplasias , Humanos , Células Matadoras Naturais , Neoplasias/genética , Apresentação de Antígeno , Genômica , Citotoxicidade Imunológica/genética , Linhagem Celular TumoralRESUMO
Malignant transformation of cells typically involves several genetic lesions, whose combined activity gives rise to cancer1. Here we analyse 1,148 patient-derived B-cell leukaemia (B-ALL) samples, and find that individual mutations do not promote leukaemogenesis unless they converge on one single oncogenic pathway that is characteristic of the differentiation stage of transformed B cells. Mutations that are not aligned with this central oncogenic driver activate divergent pathways and subvert transformation. Oncogenic lesions in B-ALL frequently mimic signalling through cytokine receptors at the pro-B-cell stage (via activation of the signal-transduction protein STAT5)2-4 or pre-B-cell receptors in more mature cells (via activation of the protein kinase ERK)5-8. STAT5- and ERK-activating lesions are found frequently, but occur together in only around 3% of cases (P = 2.2 × 10-16). Single-cell mutation and phospho-protein analyses reveal the segregation of oncogenic STAT5 and ERK activation to competing clones. STAT5 and ERK engage opposing biochemical and transcriptional programs that are orchestrated by the transcription factors MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway comes at the expense of the principal oncogenic driver and reverses transformation. Conversely, deletion of divergent pathway components accelerates leukaemogenesis. Thus, persistence of divergent signalling pathways represents a powerful barrier to transformation, while convergence on one principal driver defines a central event in leukaemia initiation. Pharmacological reactivation of suppressed divergent circuits synergizes strongly with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to enhance treatment responses.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Transformação Celular Neoplásica , Leucemia de Células B/metabolismo , Leucemia de Células B/patologia , Transdução de Sinais , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL-selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.
Assuntos
Leucemia Megacarioblástica Aguda , Leucemia Mieloide Aguda , Linfoma de Células B , Animais , Camundongos , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Proteína bcl-X/genética , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Diferenciação Celular , ApoptoseRESUMO
BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
Assuntos
Proteína Semelhante a Receptor de Calcitonina , Doença da Artéria Coronariana , Células Endoteliais , Elementos Facilitadores Genéticos , Polimorfismo de Nucleotídeo Único , Estresse Mecânico , Humanos , Células Endoteliais/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Proteína Semelhante a Receptor de Calcitonina/genética , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Mecanotransdução Celular , Células Cultivadas , Regulação da Expressão Gênica , Ligação Proteica , Predisposição Genética para Doença , Sítios de LigaçãoRESUMO
Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Criança , Humanos , Camundongos , Animais , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Peixe-Zebra/metabolismo , Tirosina , Linhagem Celular Tumoral , Transdução de Sinais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Receptores de Antígenos de Linfócitos T/uso terapêutico , Linfócitos T/metabolismo , Recidiva , Mamíferos/metabolismoRESUMO
BACKGROUND: Acute leukemia is the most common pediatric cancer, with an incidence peak at 2-5 years of age. Despite the medical advances improving survival rates, children suffer from significant side effects of treatments as well as its high social and economic impact. The frequent prenatal origin of this developmental disease follows the two-hit carcinogenesis model established in the 70s: a first hit in prenatal life with the creation of genetic fusion lesions or aneuploidy in hematopoietic progenitor/stem cells, and usually a second hit in the pediatric age that converts the preleukemic clone into clinical leukemia. Previous research has mostly focused on postnatal environmental factors triggering the second hit. SUMMARY: There is scarce evidence on prenatal risk factors associated with the first hit. Mainly retrospective case-control studies suggested several environmental and lifestyle determinants as risk factors. If these associations could be confirmed, interventions focused on modifying prenatal factors might influence the subsequent risk of leukemia during childhood and reveal unexplored research avenues for the future. In this review, we aim to comprehensively summarize the currently available evidence on prenatal risk factors for the development of childhood leukemia. According to the findings of this review, parental age, ethnicity, maternal diet, folate intake, alcohol consumption, X-ray exposure, pesticides, perinatal infections, and fetal growth may have a significant role in the appearance of preleukemic lesions during fetal life. Other factors such as socioeconomic status, consumption of caffeinated beverages, and smoking consumption have been suggested with inconclusive evidence. Additionally, investigating the association between prenatal factors and genetic lesions associated with childhood leukemia at birth is crucial. Prospective studies evaluating the link between lifestyle factors and genetic alterations could provide indirect evidence supporting new research avenues for leukemia prevention. Maternal diet and lifestyle factors are modifiable determinants associated with adverse perinatal outcomes that could be also related to preleukemic lesions. KEY MESSAGES: Parental age, ethnicity, maternal diet, folate intake, alcohol consumption, X-ray exposure, pesticides, perinatal infections, and fetal growth may have a significant role in the appearance of preleukemic lesions during fetal life. Dedicating efforts to studying maternal lifestyle during pregnancy and its association with genetic lesions leading to childhood leukemia could lead to novel prevention strategies.
Assuntos
Leucemia , Estilo de Vida , Efeitos Tardios da Exposição Pré-Natal , Humanos , Gravidez , Fatores de Risco , Feminino , Efeitos Tardios da Exposição Pré-Natal/epidemiologia , Leucemia/epidemiologia , Leucemia/etiologia , Criança , Pré-EscolarRESUMO
Lifestyle modifications, including increased physical activity and exercise, are recommended for non-alcoholic fatty liver disease (NAFLD). Inflamed adipose tissue (AT) contributes to the progression and development of NAFLD and oxylipins such as hydroxyeicosatetraenoic acids (HETE), hydroxydocosahexanenoic acids (HDHA), prostaglandins (PEG2), and isoprostanoids (IsoP), which all may play a role in AT homeostasis and inflammation. To investigate the role of exercise without weight loss on AT and plasma oxylipin concentrations in NAFLD subjects, we conducted a 12-week randomized controlled exercise intervention. Plasma samples from 39 subjects and abdominal subcutaneous AT biopsy samples from 19 subjects were collected both at the beginning and the end of the exercise intervention. In the AT of women, a significant reduction of gene expression of hemoglobin subunits (HBB, HBA1, HBA2) was observed within the intervention group during the 12-week intervention. Their expression levels were negatively associated with VO2max and maxW. In addition, pathways involved in adipocyte morphology alterations significantly increased, whereas pathways in fat metabolism, branched-chain amino acids degradation, and oxidative phosphorylation were suppressed in the intervention group (p < 0.05). Compared to the control group, in the intervention group, the ribosome pathway was activated, but lysosome, oxidative phosphorylation, and pathways of AT modification were suppressed (p < 0.05). Most of the oxylipins (HETE, HDHA, PEG2, and IsoP) in plasma did not change during the intervention compared to the control group. 15-F2t-IsoP significantly increased in the intervention group compared to the control group (p = 0.014). However, this oxylipin could not be detected in all samples. Exercise intervention without weight loss may influence the AT morphology and fat metabolism at the gene expression level in female NAFLD subjects.
Assuntos
Treinamento Intervalado de Alta Intensidade , Hepatopatia Gordurosa não Alcoólica , Humanos , Feminino , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/terapia , Hepatopatia Gordurosa não Alcoólica/complicações , Tecido Adiposo/metabolismo , Redução de Peso , Expressão Gênica , Fígado/metabolismoRESUMO
[Figure: see text].
Assuntos
Aorta/metabolismo , Doenças da Aorta/genética , Aterosclerose/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Transcriptoma , Aorta/patologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Placa Aterosclerótica , RNA-SeqRESUMO
Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Células Germinativas/metabolismo , Humanos , Linfoma/genética , Mutação , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Existing large gene expression data repositories hold enormous potential to elucidate disease mechanisms, characterize changes in cellular pathways, and to stratify patients based on molecular profiles. To achieve this goal, integrative resources and tools are needed that allow comparison of results across datasets and data types. We propose an intuitive approach for data-driven stratifications of molecular profiles and benchmark our methodology using the dimensionality reduction algorithm t-distributed stochastic neighbor embedding (t-SNE) with multi-study and multi-platform data on hematological malignancies. Our approach enables assessing the contribution of biological versus technical variation to sample clustering, direct incorporation of additional datasets to the same low dimensional representation, comparison of molecular disease subtypes identified from separate t-SNE representations, and characterization of the obtained clusters based on pathway databases and additional data. In this manner, we performed an integrative analysis across multi-omics acute myeloid leukemia studies. Our approach indicated new molecular subtypes with differential survival and drug responsiveness among samples lacking fusion genes, including a novel myelodysplastic syndrome-like cluster and a cluster characterized with CEBPA mutations and differential activity of the S-adenosylmethionine-dependent DNA methylation pathway. In summary, integration across multiple studies can help to identify novel molecular disease subtypes and generate insight into disease biology.
Assuntos
Análise por Conglomerados , Biologia Computacional/métodos , Mineração de Dados/métodos , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Fenótipo , Algoritmos , Bases de Dados Genéticas , Genes Neoplásicos , Humanos , Leucemia Mieloide Aguda/classificação , Mutação , Tamanho da AmostraRESUMO
While the shelterin complex guards and coordinates the mechanism of telomere regulation, deregulation of this process is tightly linked to malignant transformation and cancer. Here, we present the novel finding of a germline stop-gain variant (p.Q199*) in the shelterin complex gene POT1, which was identified in a child with acute myeloid leukemia. We show that the cells overexpressing the mutated POT1 display increased DNA damage and chromosomal instabilities compared to the wildtype counterpart. Protein and mRNA expression analyses in the primary patient cells further confirm that, physiologically, the variant leads to a nonfunctional POT1 allele in the patient. Subsequent telomere length measurements in the primary cells carrying heterozygous POT1 p.Q199* as well as POT1 knockdown AML cells revealed telomeric elongation as the main functional effect. These results show a connection between POT1 p.Q199* and telomeric dysregulation and highlight POT1 germline deficiency as a predisposition to myeloid malignancies in childhood.
Assuntos
Predisposição Genética para Doença/genética , Leucemia Mieloide Aguda/genética , Transtornos Mieloproliferativos/genética , Complexo Shelterina/genética , Proteínas de Ligação a Telômeros/genética , Adulto , Dano ao DNA/genética , Células Germinativas , Mutação em Linhagem Germinativa/genética , Células HEK293 , Humanos , Células Mieloides , RNA Mensageiro/genética , Telômero/genética , Adulto JovemRESUMO
Endothelial cells (ECs) differentiate from mesodermal progenitors during vasculogenesis. By comparing changes in chromatin interactions between human umbilical vein ECs, embryonic stem cells and mesendoderm cells, we identified regions exhibiting EC-specific compartmentalization and changes in the degree of connectivity within topologically associated domains (TADs). These regions were characterized by EC-specific transcription, binding of lineage-determining transcription factors and cohesin. In addition, we identified 1200 EC-specific long-range interactions (LRIs) between TADs. Most of the LRIs were connected between regions enriched for H3K9me3 involving pericentromeric regions, suggesting their involvement in establishing compartmentalization of heterochromatin during differentiation. Second, we provide evidence that EC-specific LRIs correlate with changes in the hierarchy of chromatin aggregation. Despite these rearrangements, the majority of chromatin domains fall within a pre-established hierarchy conserved throughout differentiation. Finally, we investigated the effect of hypoxia on chromatin organization. Although hypoxia altered the expression of hundreds of genes, minimal effect on chromatin organization was seen. Nevertheless, 70% of hypoxia-inducible genes situated within a TAD bound by HIF1α suggesting that transcriptional responses to hypoxia largely depend on pre-existing chromatin organization. Collectively our results show that large structural rearrangements establish chromatin architecture required for functional endothelium and this architecture remains largely unchanged in response to hypoxia.
Assuntos
Cromatina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Hipóxia Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Epigênese Genética , Heterocromatina , Humanos , Transcrição Gênica , CoesinasRESUMO
Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19+/CD20+-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Loci Gênicos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Changes in mature microRNA (miRNA) levels that occur downstream of signaling cascades play an important role during human development and disease. However, the regulation of primary microRNA (pri-miRNA) genes remains to be dissected in detail. To address this, we followed a data-driven approach and developed a transcript identification, validation and quantification pipeline for characterizing the regulatory domains of pri-miRNAs. Integration of 92 nascent transcriptomes and multilevel data from cells arising from ecto-, endo- and mesoderm lineages reveals cell type-specific expression patterns, allows fine-resolution mapping of transcription start sites (TSS) and identification of candidate regulatory regions. We show that inter- and intragenic pri-miRNA transcripts span vast genomic regions and active TSS locations differ across cell types, exemplified by the mir-29aâ¼29b-1, mir-100â¼let-7a-2â¼125b-1 and miR-221â¼222 clusters. Considering the presence of multiple TSS as an important regulatory feature at miRNA loci, we developed a strategy to quantify differential TSS usage. We demonstrate that the TSS activities associate with cell type-specific super-enhancers, differential stimulus responsiveness and higher-order chromatin structure. These results pave the way for building detailed regulatory maps of miRNA loci.
Assuntos
Cromatina/química , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , MicroRNAs/genética , Transcriptoma , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Ectoderma/citologia , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Endoderma/citologia , Endoderma/crescimento & desenvolvimento , Endoderma/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Família Multigênica , Especificidade de Órgãos , Regiões Promotoras Genéticas , Sítio de Iniciação de TranscriçãoRESUMO
Type 2 diabetes mellitus (T2DM) is a major risk factor for heart disease. Mortality rates after myocardial infarction (MI) are significantly increased in T2DM patients because of dysfunctional left ventricle (LV). However, molecular pathways underlying accelerated heart failure (HF) after MI in T2DM remain unclear. We investigated the underlying mechanisms by inducing MI in a well-established model of T2DM and control mice. Cardiac imaging revealed a significantly decreased global left ventricular ejection fraction in parallel with increased mortality after MI in T2DM mice compared with control mice. Genome-wide mRNA sequencing, immunoblot, electron microscopy, together with immunofluorescence staining for LC3 and p62 indicated an impaired mitophagy in peri-infarct regions of LV in T2DM mice compared with control mice. Furthermore, defective mitophagy was associated with an increased release of mitochondrial DNA, resulting in Aim2 and NLRC4 inflammasome and caspase-I hyperactivation in cardiomyocytes and cardiac macrophages in peri-infarct regions of LV in T2DM mice. Consistent with inflammasome and caspase-I hyperactivation, cardiomyocyte death and IL-18 secretion were increased in T2DM mice. Our results indicate that T2DM aggravates HF after MI through defective mitophagy, associated exaggerated inflammasome activation, cell death, and IL-18 secretion, suggesting that restoring mitophagy and inhibiting inflammasome activation may serve as novel targets for the prevention and treatment of HF in T2DM.
Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Insuficiência Cardíaca/fisiopatologia , Inflamassomos/metabolismo , Mitofagia/fisiologia , Animais , Feminino , Insuficiência Cardíaca/etiologia , Masculino , Camundongos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologiaRESUMO
Transcription factor binding specificity is crucial for proper target gene regulation. Motif discovery algorithms identify the main features of the binding patterns, but the accuracy on the lower affinity sites is often poor. Nuclear factor E2-related factor 2 (NRF2) is a ubiquitous redox-activated transcription factor having a key protective role against endogenous and exogenous oxidant and electrophile stress. Herein, we decipher the effects of sequence variation on the DNA binding sequence of NRF2, in order to identify both genome-wide binding sites for NRF2 and disease-associated regulatory SNPs (rSNPs) with drastic effects on NRF2 binding. Interactions between NRF2 and DNA were studied using molecular modelling, and NRF2 chromatin immunoprecipitation-sequence datasets together with protein binding microarray measurements were utilized to study binding sequence variation in detail. The binding model thus generated was used to identify genome-wide binding sites for NRF2, and genomic binding sites with rSNPs that have strong effects on NRF2 binding and reside on active regulatory elements in human cells. As a proof of concept, miR-126-3p and -5p were identified as NRF2 target microRNAs, and a rSNP (rs113067944) residing on NRF2 target gene (Ferritin, light polypeptide, FTL) promoter was experimentally verified to decrease NRF2 binding and result in decreased transcriptional activity.
Assuntos
Genoma Humano , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Transcrição Gênica , Algoritmos , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Methodological advances that allow deeper characterization of non-coding elements in the genome have started to reveal the full spectrum of deregulation in cancer. We generated an inducible cell model to track transcriptional changes after induction of a well-known leukemia-inducing fusion gene, ETV6-RUNX1. Our data revealed widespread transcriptional alterations outside coding elements in the genome. This adds to the growing list of various alterations in the non-coding genome in cancer and pinpoints their role in diseased cellular state.
Assuntos
Regulação Leucêmica da Expressão Gênica , Genoma , Leucemia/genética , RNA não Traduzido/genética , Humanos , Proteínas de Fusão Oncogênica/metabolismo , RNA não Traduzido/metabolismoRESUMO
KEY POINTS: Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart. ABSTRACT: The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.
Assuntos
Coração/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Animais , Sinalização do Cálcio , Masculino , Camundongos Transgênicos , Contração Miocárdica , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , FenótipoRESUMO
The distinct cell types of multicellular organisms arise owing to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We curated human expression data comprising 166 cell types and 2,602 transcription-regulating genes and developed a data-driven method for identifying putative determinants of cell fate built around the concept of expression reversal of gene pairs, such as those participating in toggle-switch circuits. This approach allows us to organize the cell types into their ontogenic lineage relationships. Our method identifies genes in regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, and it may be useful for prioritizing candidate factors for direct conversion of cell fate.
Assuntos
Linhagem da Célula , Redes Reguladoras de Genes , Transcriptoma , Diferenciação Celular , HumanosRESUMO
Metabolic diseases and comorbidities represent an ever-growing epidemic where multiple cell types impact tissue homeostasis. Here, the link between the metabolic and gene regulatory networks was studied through experimental and computational analysis. Integrating gene regulation data with a human metabolic network prompted the establishment of an open-sourced web portal, IDARE (Integrated Data Nodes of Regulation), for visualizing various gene-related data in context of metabolic pathways. Motivated by increasing availability of deep sequencing studies, we obtained ChIP-seq data from widely studied human umbilical vein endothelial cells. Interestingly, we found that association of metabolic genes with multiple transcription factors (TFs) enriched disease-associated genes. To demonstrate further extensions enabled by examining these networks together, constraint-based modeling was applied to data from human preadipocyte differentiation. In parallel, data on gene expression, genome-wide ChIP-seq profiles for peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding protein (CEBP) α, liver X receptor (LXR) and H3K4me3 and microRNA target identification for miR-27a, miR-29a and miR-222 were collected. Disease-relevant key nodes, including mitochondrial glycerol-3-phosphate acyltransferase (GPAM), were exposed from metabolic pathways predicted to change activity by focusing on association with multiple regulators. In both cell types, our analysis reveals the convergence of microRNAs and TFs within the branched chain amino acid (BCAA) metabolic pathway, possibly providing an explanation for its downregulation in obese and diabetic conditions.