RESUMO
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
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
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
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
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
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 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 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 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
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
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
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
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
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
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Transcrição GATA2/metabolismo , Hematopoese , Proteínas de Neoplasias/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Sequência Conservada , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Alinhamento de Sequência , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de TempoRESUMO
The endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed -5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of -5.5HS coupled to either PP or the SV40 promoter were able to direct beta-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The -5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell-specific transcriptional regulation that reflects the common origin of these cell types.
Assuntos
Região 5'-Flanqueadora/genética , Antígenos CD/genética , Células Endoteliais/fisiologia , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Receptores de Superfície Celular/genética , Transcrição Gênica/genética , Animais , Antígenos CD/biossíntese , Células Endoteliais/citologia , Receptor de Proteína C Endotelial , Células HeLa , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Receptores de Superfície Celular/biossíntese , Transgenes/genética , Células U937RESUMO
Transcriptional control has been identified as a key mechanism regulating the formation and subsequent behavior of hematopoietic stem cells. We have used a comparative genomics approach to identify transcriptional regulatory elements of the LMO2 gene, a transcriptional cofactor originally identified through its involvement in T-cell leukemia and subsequently shown to be critical for normal hematopoietic and endothelial development. Of the 2 previously characterized LMO2 promoters, the second (proximal) promoter was highly conserved in vertebrates ranging from mammals to fish. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis identified this promoter as the predominant source of transcription in hematopoietic tissue. Transient and stable transfections indicated that the proximal promoter was active in hematopoietic progenitor and endothelial cell lines and this activity was shown to depend on 3 conserved Ets sites that were bound in vivo by E74-like factor 1 (Elf1), Friend leukemia integration 1 (Fli1), and erythroblastosis virus oncogene homolog E twenty-six-1 (Ets1). Finally, transgenic analysis demonstrated that the LMO2 proximal promoter is sufficient for expression in endothelial cells in vivo. No hematopoietic expression was observed, indicating that additional enhancers are required to mediate transcription from the proximal promoter in hematopoietic cells. Together, these results suggest that the conserved proximal promoter is central to LMO2 transcription in hematopoietic and endothelial cells, where it is regulated by Ets factors.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Efrina-A2/metabolismo , Regulação da Expressão Gênica/genética , Metaloproteínas/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Conservada/genética , Cães , Efrina-A2/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas com Domínio LIM , Camundongos , Dados de Sequência Molecular , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-fli-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Ratos , Elementos de Resposta/genética , Alinhamento de Sequência , Transativadores/genética , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica/genéticaRESUMO
The development of blood has long served as a model for mammalian cell type specification and differentiation, and yet the underlying transcriptional networks remain ill defined. Characterization of such networks will require genome-wide identification of cis-regulatory sequences and an understanding of how regulatory information is encoded in the primary DNA sequence. Despite progress in lower organisms, genome-wide computational identification of mammalian cis-regulatory sequences has been hindered by increased genomic complexity and cumbersome transgenic assays. Starting with a well-characterized blood stem cell enhancer from the SCL gene, we have developed computational tools for the identification of functionally related gene regulatory sequences. Two candidate enhancers discovered in this way were located in intron 1 of the Fli-1 and PRH/Hex genes, both transcription factors previously implicated in controlling blood and endothelial development. Subsequent transgenic and biochemical analysis demonstrated that the two computationally identified enhancers are functionally related to the SCL stem cell enhancer. The approach developed here may therefore be useful for identifying additional enhancers involved in the control of early blood and endothelial development, and may be adapted to decipher transcriptional regulatory codes controlling a broad range of mammalian developmental programmes.
Assuntos
Diferenciação Celular/genética , Endotélio/fisiologia , Elementos Facilitadores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Antígenos CD34/genética , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endotélio/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Proteína Proto-Oncogênica c-fli-1 , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Software , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
We previously reported that the long terminal repeats (LTRs) of retroviral elements belonging to the HERV-E family contribute to the expression of the human apolipoprotein C1 (APOC1) and endothelin B receptor (EDNRB) genes by providing alternative promoters. While both LTRs were shown to promote transcription in vivo and in vitro, their respective activity and tissue specificity appeared to differ even though they shared a high degree of sequence identity. In the present study, we further characterized the promoter of the EDNRB LTR and delineated the regions and motifs required for strong activity. We confirmed the placenta-restricted expression of the LTR by transient transfections and quantitative real-time PCR and determined that the retroviral promoter contributes significantly to the level of EDNRB transcripts in placenta, where chimeric mRNAs were found to represent 15% of overall EDNRB mRNAs. Transient transfection of 5' deletion constructs in cells of placental origin identified a motif, named LPE1, between positions 111 and 122 of the EDNRB LTR necessary for transcriptional activity. Removal of this region, which contains a putative SP1 binding site, abolished promoter activity. A second enhancing region resides between positions 175 and 215 of the LTR and was termed LPE2. Interestingly, this section contained three binding sites that were not present in the APOC1 LTR due to minor nucleotide differences. The predicted motifs in the EDNRB LTR were found to likely act in symbiosis as modifications to any of the three sites reduced transcription by one-third while alterations to all three eliminated promoter activity. The results from this study illustrate how slight variations in transcriptional regulatory sequences can have a profound effect on promoter activity and demonstrate the complex regulatory effects of human endogenous retrovirus elements on human gene expression.