RESUMO
Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.
Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberrações Cromossômicas , Citogenética , Fusão Gênica , Rearranjo Gênico/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imuno-Histoquímica , Recém-Nascido , Leucemia Mieloide Aguda/genética , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.
Assuntos
Ribonuclease III/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Regiões Terminadoras Genéticas/fisiologia , Transcrição Gênica/fisiologia , Actinas/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Álcool Desidrogenase/genética , DNA/metabolismo , Exorribonucleases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Poliadenilação/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Inativação Gênica/imunologia , Células Matadoras Naturais/citologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Subfamília D de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias/genética , Neoplasias/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transexualidade/genéticaRESUMO
The molecular mechanisms regulating self-renewal of leukemia stem cells remain poorly understood. Here we report the generation of 2 closely related leukemias created through the retroviral overexpression of Meis1 and Hoxa9. Despite their apparent common origin, these clonal leukemias exhibit enormous differences in stem cell frequency (from 1 in 1.4, FLA2; to 1 in 347, FLB1), suggesting that one of these leukemias undergoes nearly unlimited self-renewal divisions. Using next-generation RNA-sequencing, we characterized the transcriptomes of these phenotypically similar, but biologically distinct, leukemias, identifying hundreds of differentially expressed genes and a large number of structural differences (eg, alternative splicing and promoter usage). Focusing on ligand-receptor pairs, we observed high expression levels of Sdf1-Cxcr4; Jagged2-Notch2/1; Osm-Gp130; Scf-cKit; and Bmp15-Tgfb1/2. Interestingly, the integrin beta 2-like gene (Itgb2l) is both highly expressed and differentially expressed between our 2 leukemias (â¼ 14-fold higher in FLA2 than FLB1). In addition, gene ontology analysis indicated G-protein-coupled receptor had a much higher proportion of differential expression (22%) compared with other classes (â¼ 5%), suggesting a potential role regulating subtle changes in cellular behavior. These results provide the first comprehensive transcriptome analysis of a leukemia stem cell and document an unexpected level of transcriptome variation between phenotypically similar leukemic cells.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas , Animais , Células Clonais , Citometria de Fluxo , Vetores Genéticos , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Proteína Meis1 , Proteínas de Neoplasias/genética , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
Ets transcription factors play important roles during the development and maintenance of the haematopoietic system. One such factor, Elf-1 (E74-like factor 1) controls the expression of multiple essential haematopoietic regulators including Scl/Tal1, Lmo2 and PU.1. However, to integrate Elf-1 into the wider regulatory hierarchies controlling haematopoietic development and differentiation, regulatory elements as well as upstream regulators of Elf-1 need to be identified. Here, we have used locus-wide comparative genomic analysis coupled with chromatin immunoprecipitation (ChIP-chip) assays which resulted in the identification of five distinct regulatory regions directing expression of Elf-1. Further, ChIP-chip assays followed by functional validation demonstrated that the key haematopoietic transcription factor PU.1 is a major upstream regulator of Elf-1. Finally, overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated that Elf-1 downregulation is necessary for terminal erythroid differentiation. Given the known activation of PU.1 by Elf-1 and our newly identified reciprocal activation of Elf-1 by PU.1, identification of an inhibitory role for Elf-1 has significant implications for our understanding of how PU.1 controls myeloid-erythroid differentiation. Our findings therefore not only represent the first report of Elf-1 regulation but also enhance our understanding of the wider regulatory networks that control haematopoiesis.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Células Eritroides/citologia , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Acetilação , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Células Eritroides/metabolismo , Loci Gênicos , Genômica , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição GATA/metabolismo , Leucemia/metabolismo , Metaloproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Telomerase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma/genética , Proteínas com Domínio LIM , Leucemia/genética , Metaloproteínas/genética , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Telomerase/genética , Análise Serial de Tecidos , Transativadores/genéticaRESUMO
Posttransplant leukemia detection before overt relapse is key to the success of immunotherapeutic interventions, as they are more efficient when leukemia burden is low. However, optimal schedule and monitoring methods are not well defined. We report the intensive bone marrow monitoring of minimal residual disease (MRD) using flow cytometry (FC) and nested reverse transcription polymerase chain reaction (RT-PCR) whenever a fusion transcript allowed it and chimerism by PCR at 11 timepoints in the first 2 years after transplant. Seventy-one transplants were performed in 59 consecutive children, for acute myeloid (n = 38), lymphoid (n = 31), or mixed-phenotype (n = 2) leukemia. MRD was monitored in 62 cases using FC (n = 58) and/or RT-PCR (n = 35). Sixty-seven percent of leukemia recurrences were detected before overt relapse, with a detection rate of 89% by RT-PCR and 40% by FC alone. Increased mixed chimerism was never the first evidence of recurrence. Two patients monitored by RT-PCR relapsed without previous MRD detection, one after missed scheduled evaluation and the other 4.7 years post transplant. Among the 22 cases with MRD detection without overt relapse, 19 received therapeutic interventions. Eight (42%) never relapsed. In conclusion, intensive marrow monitoring by RT-PCR effectively allows for early detection of posttransplant leukemia recurrence.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Quimerismo , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Recidiva , Transplante HomólogoRESUMO
Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.
Assuntos
Antígenos CD/genética , Sangue/metabolismo , Endotélio/metabolismo , Fatores de Transcrição GATA/fisiologia , Hemangioblastos/fisiologia , Proteína Proto-Oncogênica c-ets-1/fisiologia , Receptores de Superfície Celular/genética , Animais , Antígenos CD/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Endoglina , Fatores de Transcrição GATA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hemangioblastos/metabolismo , Sistema Hematopoético/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
The ability to quantitatively survey the global behavior of transcriptomes has been a key milestone in the field of systems biology, enabled by the advent of DNA microarrays. While this approach has literally transformed our vision and approach to cellular physiology, microarray technology has always been limited by the requirement to decide, a priori, what regions of the genome to examine. While very high density tiling arrays have reduced this limitation for simpler organisms, it remains an obstacle for larger, more complex, eukaryotic genomes. The recent development of "next-generation" massively parallel sequencing (MPS) technologies by companies such as Roche (454 GS FLX), Illumina (Genome Analyzer II), and ABI (AB SOLiD) has completely transformed the way in which quantitative transcriptomics can be done. These new technologies have reduced both the cost-per-reaction and time required by orders of magnitude, making the use of sequencing a cost-effective option for many experimental approaches. One such method that has recently been developed uses MPS technology to directly survey the RNA content of cells, without requiring any of the traditional cloning associated with EST sequencing. This approach, called "RNA-seq", can generate quantitative expression scores that are comparable to microarrays, with the added benefit that the entire transcriptome is surveyed without the requirement of a priori knowledge of transcribed regions. The important advantage of this technique is that not only can quantitative expression measures be made, but transcript structures including alternatively spliced transcript isoforms, can also be identified. This article discusses the experimental approach for both sample preparation and data analysis for the technique of RNA-seq.
Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , HumanosRESUMO
Shwachman-Diamond syndrome (SDS) is a rare and systemic disease mostly caused by mutations in the SBDS gene and characterized by pancreatic insufficiency, skeletal abnormalities, and a bone marrow dysfunction. In addition, SDS patients are predisposed to develop myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), typically during adulthood and associated with TP53 mutations. Although most SDS diagnoses are established in childhood, the nature and frequency of serial bone marrow cell investigations during the patients' lifetime remain a debatable topic. The precise molecular mechanisms leading to AML progression in SDS patients have not been fully elucidated because the patient cohorts are small and most disease monitoring is conducted using standard histological and cytogenetic approaches. Here we report a rare case of a patient with SDS who was diagnosed with AML at 5 years of age and survived. Intermittent neutropenia preceded the AML diagnostic but serial bone marrow monitoring according to the standard of care revealed no cytogenetic anomalies nor signs of clonal hematopoiesis. Using next generation sequencing approaches to find cytogenetically cryptic pathogenic mutations, we identified the cancer hotspot mutation c.394C>T/p.Arg132Cys in IDH1 with high variant allelic frequency in bone marrow cells, suggesting clonal expansion of a major leukemic clone karyotypically normal, in the SDS-associated AML. The mutation was somatic and likely occurred at the leukemic transformation stage, as it was not detected in a matched normal tissue nor in bone marrow smear prior to AML diagnosis. Gain-of-function mutations in IDH1, such as c.394C>T/p.Arg132Cys, create a neo-activity of isocitrate dehydrogenase 1 converting α-ketoglutarate into the oncometabolite D-2-hydroxyglutarate, inhibiting α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Overall, our results suggest that along with previously described abnormalities such as TP53 mutations or monosomy7, 7q-, which are all absent in this patient, additional mechanisms including IDH1 mutations drive SDS-related AML and are likely associated with variable outcomes. Sensitive techniques complementary to standard cytogenetics, such as unbiased or targeted panel-based next generation sequencing approaches, warrant testing for monitoring of myelodysplasia, clonal hematopoiesis, and leukemia in the context SDS. Such analyses would also assist treatment decisions and allow to gain insight into the disease biology.
RESUMO
Acute megakaryoblastic leukemia (AMKL) represents â¼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.
Assuntos
Biomarcadores , Modelos Animais de Doenças , Leucemia Megacarioblástica Aguda/etiologia , Leucemia Megacarioblástica Aguda/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional/métodos , Suscetibilidade a Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Leucemia Megacarioblástica Aguda/terapia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nearly half of mammalian genomes are derived from ancient transposable elements (TEs). We analyzed the prevalence of TEs in untranslated regions of human and mouse mRNAs and found evidence suggesting that TEs affect the expression of many genes through the donation of transcriptional regulatory signals. Furthermore, we found that recently expanded gene classes, such as those involved in immunity or response to external stimuli, have transcripts enriched in TEs, whereas TEs are excluded from mRNAs of highly conserved genes with basic functions in development or metabolism. These results support the view that TEs have played a significant role in the diversification and evolution of mammalian genes.
Assuntos
Elementos de DNA Transponíveis , Evolução Molecular , Variação Genética , Animais , Aromatase/genética , Aromatase/metabolismo , Humanos , Camundongos , Regiões Promotoras GenéticasRESUMO
Product of the Itga2b gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, Itga2b can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in Itga2b regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the Mpl locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that Itga2b expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the Itga2b Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both Itga2b and Mpl expression.
Assuntos
Hematopoese/fisiologia , Integrina alfa2/metabolismo , Receptores de Trombopoetina/biossíntese , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Imunoprecipitação da Cromatina , Análise por Conglomerados , DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Hematopoese/genética , Humanos , Megacariócitos/citologia , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismoRESUMO
The ETS transcription factor Elf-4 is an important regulator of hematopoietic stem cell (HSC) and T cell homeostasis. To gain insights into the transcriptional circuitry within which Elf-4 operates, we used comparative sequence analysis coupled with chromatin immunoprecipitation (ChIP) with microarray technology (ChIP-chip) assays for specific chromatin marks to identify three promoters and two enhancers active in hematopoietic and endothelial cell lines. Comprehensive functional validation of each of these regulatory regions in transgenic mouse embryos identified a tissue-specific enhancer (-10E) that displayed activity in fetal liver, dorsal aorta, vitelline vessels, yolk sac, and heart. Integration of a ChIP-sequencing (ChIP-Seq) data set for 10 key stem cell transcription factors showed Pu.1, Fli-1, and Erg were bound to the -10E element, and mutation of three highly conserved ETS sites within the enhancer abolished its activity. Finally, the transcriptional repressor Gfi1b was found to bind to and repress one of the Elf-4 promoters (-30P), and we show that this repression of Elf-4 is important for the maturation of primary fetal liver erythroid cells. Taken together, our results provide a comprehensive overview of the transcriptional control of Elf-4 within the hematopoietic system and, thus, integrate Elf-4 into the wider transcriptional regulatory networks that govern hematopoietic development.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Sequência Conservada , DNA/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Eritropoese/genética , Redes Reguladoras de Genes , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido NucleicoRESUMO
The growth factor independence 1 (Gfi1) gene was originally discovered in the hematopoietic system, where it functions as a key regulator of stem cell homeostasis, as well as neutrophil and T-cell development. Outside the blood system, Gfi1 is essential for inner-ear hair and intestinal secretory cell differentiation. To understand the regulatory hierarchies within which Gfi1 operates to control these diverse biological functions, we used a combination of comparative genomics, locus-wide chromatin immunoprecipitation assays, functional validation in cell lines, and extensive transgenic mouse assays to identify and characterize the complete ensemble of Gfi1 regulatory elements. This concerted effort identified five distinct regulatory elements spread over 100kb each driving expression in transgenic mice to a subdomain of endogenous Gfi1. Detailed characterization of an enhancer 35 kb upstream of Gfi1 demonstrated activity in the dorsal aorta region and fetal liver in transgenic mice, which was bound by key stem cell transcription factors Scl/Tal1, PU.1/Sfpi1, Runx1, Erg, Meis1, and Gata2. Taken together, our results reveal the regulatory regions responsible for Gfi1 expression and importantly establish that Gfi1 expression at the sites of hematopoietic stem cell (HSC) emergence is controlled by key HSC regulators, thus integrating Gfi1 into the wider HSC regulatory networks.
Assuntos
Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Sistema Hematopoético/metabolismo , Proteínas de Homeodomínio , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas , Sequências Reguladoras de Ácido Nucleico , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Transcription factors such as Scl/Tal1, Lmo2, and Runx1 are essential for the development of hematopoietic stem cells (HSCs). However, the precise mechanisms by which these factors interact to form transcriptional networks, as well as the identity of the genes downstream of these regulatory cascades, remain largely unknown. To this end, we generated an Scl(-/-) yolk sac cell line to identify candidate Scl target genes by global expression profiling after reintroduction of a TAT-Scl fusion protein. Bioinformatics analysis resulted in the identification of 9 candidate Scl target transcription factor genes, including Runx1 and Runx3. Chromatin immunoprecipitation confirmed that both Runx genes are direct targets of Scl in the fetal liver and that Runx1 is also occupied by Scl in the yolk sac. Furthermore, binding of an Scl-Lmo2-Gata2 complex was demonstrated to occur on the regions flanking the conserved E-boxes of the Runx1 loci and was shown to transactivate the Runx1 element. Together, our data provide a key component of the transcriptional network of early hematopoiesis by identifying downstream targets of Scl that can explain key aspects of the early Scl(-/-) phenotype.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Fígado/embriologia , Fígado/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Saco Vitelino/embriologia , Saco Vitelino/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Separação Celular , Sequência Conservada , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas com Domínio LIM , Metaloproteínas/genética , Metaloproteínas/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1, and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. Smad6, Bmp4, and Runx1 transcripts are concentrated along the ventral aspect of the E10.5 dorsal aorta in the aorta-gonad-mesonephros region from which HSCs originate. Moreover, Smad6, an inhibitor of Bmp4 signaling, binds and inhibits Runx1 activity, whereas Smad1, a positive mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl transcriptional network, Runx1 activity, and the Bmp4/Smad signaling pathway.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Animais , Sequência de Bases , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Biologia Computacional , Sequência Conservada , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Família Multigênica , Células NIH 3T3 , Ligação Proteica , Elementos Reguladores de Transcrição/genética , Alinhamento de Sequência , Proteína Smad6/química , Proteína Smad6/genética , Proteína Smad6/metabolismoRESUMO
Conservation of the vertebrate body plan has been attributed to the evolutionary stability of gene-regulatory networks (GRNs). We describe a regulatory circuit made up of Gata2, Fli1, and Scl/Tal1 and their enhancers, Gata2-3, Fli1+12, and Scl+19, that operates during specification of hematopoiesis in the mouse embryo. We show that the Fli1+12 enhancer, like the Gata2-3 and Scl+19 enhancers, targets hematopoietic stem cells (HSCs) and relies on a combination of Ets, Gata, and E-Box motifs. We show that the Gata2-3 enhancer also uses a similar cluster of motifs and that Gata2, Fli1, and Scl are expressed in embryonic day-11.5 dorsal aorta where HSCs originate and in fetal liver where they multiply. The three HSC enhancers in these tissues and in ES cell-derived hemangioblast equivalents are bound by each of these transcription factors (TFs) and form a fully connected triad that constitutes a previously undescribed example of both this network motif in mammalian development and a GRN kernel operating during the specification of a mammalian stem cell.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Transcrição GATA2/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/fisiologia , Embrião de Mamíferos/fisiologia , Elementos Facilitadores Genéticos , Fator de Transcrição GATA2/química , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica , Camundongos , Proteína Proto-Oncogênica c-fli-1/química , Proteína Proto-Oncogênica c-fli-1/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Transcription factors are key regulators of hematopoietic stem cells (HSCs), yet the molecular mechanisms that control their expression are largely unknown. Previously, we demonstrated that expression of Scl/Tal1, a transcription factor required for the specification of HSCs, is controlled by Ets and GATA factors. Here we characterize the molecular mechanisms controlling expression of Lyl1, a paralog of Scl also required for HSC function. Two closely spaced promoters directed expression to hematopoietic progenitor, megakaryocytic, and endothelial cells in transgenic mice. Conserved binding sites required for promoter activity were bound in vivo by GATA-2 and the Ets factors Fli1, Elf1, Erg, and PU.1. However, despite coregulation of Scl and Lyl1 by the same Ets and GATA factors, Scl expression was initiated prior to Lyl1 in embryonic stem (ES) cell differentiation assays. Moreover, ectopic expression of Scl but not Lyl1 rescued hematopoietic differentiation in Scl-/- ES cells, thus providing a molecular explanation for the vastly different phenotypes of Scl-/- and Lyl1-/- mouse embryos. Furthermore, coregulation of Scl and Lyl1 later during development may explain the mild phenotype of Scl-/- adult HSCs.