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COVID-19/virología , Receptores Virales/genética , SARS-CoV-2/genética , Factores de Transcripción/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , COVID-19/genética , COVID-19/metabolismo , Modelos Animales de Enfermedad , Factores de Unión al ADN Específico de las Células Eritroides/genética , Factor Nuclear 4 del Hepatocito/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Intestinos/virología , Ratones , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Proteína Smad4/genética , Glicoproteína de la Espiga del Coronavirus/genética , Factores de Transcripción/metabolismo , Internalización del VirusRESUMEN
Mutations of the Gon4l/udu gene in different organisms give rise to diverse phenotypes. Although the effects of Gon4l/Udu in transcriptional regulation have been demonstrated, they cannot solely explain the observed characteristics among species. To further understand the function of Gon4l/Udu, we used yeast two-hybrid (Y2H) screening to identify interacting proteins in zebrafish and mouse systems, confirmed the interactions by co-immunoprecipitation assay, and found four novel Gon4l-interacting proteins: BRCA1 associated protein-1 (Bap1), DNA methyltransferase 1 (Dnmt1), Tho complex 1 (Thoc1, also known as Tho1 or HPR1), and Cryptochrome circadian regulator 3a (Cry3a). Furthermore, all known Gon4l/Udu-interacting proteins-as found in this study, in previous reports, and in online resources-were investigated by Phenotype Enrichment Analysis. The most enriched phenotypes identified include increased embryonic tissue cell apoptosis, embryonic lethality, increased T cell derived lymphoma incidence, decreased cell proliferation, chromosome instability, and abnormal dopamine level, characteristics that largely resemble those observed in reported Gon4l/udu mutant animals. Similar to the expression pattern of udu, those of bap1, dnmt1, thoc1, and cry3a are also found in the brain region and other tissues. Thus, these findings indicate novel mechanisms of Gon4l/Udu in regulating CpG methylation, histone expression/modification, DNA repair/genomic stability, and RNA binding/processing/export.
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Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Ratones , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina Tiolesterasa/metabolismo , Pez CebraRESUMEN
Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.
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Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Corazón/embriología , Miocitos Cardíacos/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Cromatina/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Atrios Cardíacos/embriología , Atrios Cardíacos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/fisiología , Fase S/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiologíaRESUMEN
Anteroposterior (AP) axis extension during gastrulation requires embryonic patterning and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation through direct positive and negative regulation of gene expression. Although identified as a recessive enhancer of impaired axis extension in planar cell polarity (PCP) mutants, udu functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. Gon4l limits expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrinα3b, excesses of which perturb the notochord boundary via tension-dependent and -independent mechanisms, respectively. By promoting formation of this AP-aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis along the AP embryonic axis.
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Factores de Unión al ADN Específico de las Células Eritroides/genética , Factores de Unión al ADN Específico de las Células Eritroides/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Animales , Tipificación del Cuerpo , Adhesión Celular , Comunicación Celular , Cromatina/química , Cruzamientos Genéticos , Glicoproteínas de Membrana/fisiología , Mutación , Notocorda/fisiología , Análisis de Secuencia de ARN , Transducción de Señal , Xenopus , Pez CebraRESUMEN
Confirming the binding of a transcription factor with a particular DNA sequence may be important in characterizing interactions with a synthetic promoter. Electrophoretic mobility shift assay is a powerful approach to demonstrate the specific DNA sequence that is bound by a transcription factor and also to confirm the specific transcription factor involved in the interaction. In this chapter we describe a method we have successfully used to demonstrate interactions of endogenous transcription factors with sequences derived from endogenous and synthetic promoters.
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ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética/métodos , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , ADN/química , ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Humanos , Células Jurkat , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
OBJETIVOS: Verificar se a presença de agentes infecciosos no conteúdo vaginal ou cervical pode alterar os resultados dos testes da proteína-1 fosforilada ligada ao fator de crescimento insulina-símile (phIGFBP-1) e das medidas do comprimento do colo uterino (CC) pela ultrassonografia transvaginal. MÉTODOS: Um total de 107 gestantes com antecedente de prematuridade espontânea foram submetidas ao teste da phIGFBP-1 e à realização da ultrassonografia transvaginal para medida do comprimento do colo uterino, a cada três semanas, entre 24 e 34 semanas. As infecções genitais foram pesquisadas imediatamente antes da realização dos testes. As pacientes foram distribuídas em quatro grupos (GA, GB, GC e GD) e dentro de cada grupo foi avaliada a correlação entre infecção genital e alteração nos testes utilizando a análise das razões de chance (OR) e o coeficiente de correlação de Pearson. RESULTADOS: Em cada grupo, mais de 50% das pacientes apresentaram infecção genital (GA 10/17; GB 28/42; GC 15/24; GD 35/53), sendo a vaginose bacteriana a principal alteração de flora vaginal. O resultado positivo para phIGFBP-1 (GA 10/10; GB 18/28; GC 15/15; GD 19/35) e CC≤20 mm (GA 10/10; GB 20/28; GC 10/15; GD 20/35) foram os resultados encontrados com maior frequência nas pacientes com infecção genital em todos os grupos. Porém, aplicando o coeficiente de correlação de Pearson foi identificada correlação entre infecção genital e positividade para os marcadores. CONCLUSÃO: A presença de alteração da flora vaginal e de outras infecções genitais não alteram significativamente os resultados do teste da phIGFBP-1 e da medida do colo uterino quando comparados aos casos sem infecção. No entanto, é necessária ...
PURPOSE: To determine if the presence of infectious agents in vaginal or cervical content can alter the results of the insulin-like growth factor binding protein-1 (phIGFBP-1) test and the measurement of cervical length (CC) by transvaginal ultrasonography. METHODS: A total of 107 pregnant women with a history of spontaneous preterm birth were submitted to the phIGFBP-1 test and to measurement of CC by transvaginal ultrasonography every 3 weeks, between 24 and 34 weeks of gestation. Genital infections were determined immediately before testing. The patients were distributed into four groups (GA, GB, GC, and GD) and the correlation between genital infection and changes in the tests was determined within each group based on the odds ratio (OR) and the Pearson correlation coefficient. RESULTS: In each group, over 50% of the patients had genital infections (GA 10/17; GB 28/42; GC 15/24; GD 35/53), with bacterial vaginosis being the main alteration of the vaginal flora. Positive results for phIGFBP-1(GA 10/10; GB 18/28; GC 15/15; GD 19/35) and CC≤20 mm (GA 10/10; GB 20/28; GC 10/15; GD 20/35) were obtained more frequently in patients with genital infection in all groups. Nonetheless, when applying the Pearson correlation coefficient we detected a poor correlation between genital infection and positivity for markers. CONCLUSION: The presence of changes in the vaginal flora and of other genital infections does not significantly alter the results of phIGFBP-1 and the measurement of cervical length when compared to cases without infection. However, more studies with larger samples are necessary to confirm these results. .
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Humanos , Antimetabolitos Antineoplásicos/farmacología , Células Precursoras Eritroides/citología , Fenilacetatos/farmacología , Factores de Transcripción/metabolismo , Antígenos de Superficie/metabolismo , Línea Celular , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Células Precursoras Eritroides/efectos de los fármacos , Citometría de Flujo , Factor de Transcripción GATA1 , Globinas/metabolismo , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and ß-major globin), and proteins (Gata-1 and ß-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.
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Alginatos/metabolismo , Diferenciación Celular/fisiología , Citocinas/metabolismo , Células Madre Embrionarias/fisiología , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Desarrollo de Músculos/fisiología , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Células Madre Embrionarias/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Ácido Glucurónico/metabolismo , Células Madre Hematopoyéticas/fisiología , Células Hep G2 , Ácidos Hexurónicos/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Interleucina-3/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Factor de Células Madre/metabolismo , Globinas beta/metabolismo , Globinas zeta/metabolismoRESUMEN
Two mechanisms that play important roles in cell fate decisions are control of a "core transcriptional network" and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.
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Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Linfocitos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Inmunoprecipitación de Cromatina , Eritrocitos , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Modelos Teóricos , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Proteína 1 de la Leucemia Linfocítica T Aguda , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
Overexpression of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)-κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti-inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit-erythroid colony formation in human CD34(+) cells. CD34(+) cells pre-incubated with resveratrol for 72 h in the presence of TNFα inhibited NF-κB activation via decreased NF-κB nuclear localization without altering total NF-κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34(+) cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα-mediated NF-κB activation and promote erythropoiesis in primary human CD34(+) cells.
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Antiinflamatorios no Esteroideos/farmacología , Eritropoyesis/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Estilbenos/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adulto , Antígenos CD34/análisis , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Células Precursoras Eritroides/efectos de los fármacos , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Humanos , Persona de Mediana Edad , FN-kappa B/metabolismo , FN-kappa B/fisiología , Resveratrol , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu(tu24) mutants. We further showed that the extensive p53-dependent apoptosis in udu(tu24) mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2'-deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity.
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Daño del ADN , Embrión no Mamífero/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/genética , Mutación , Proteínas de Pez Cebra/genética , Animales , Apoptosis/genética , Tipificación del Cuerpo/genética , Bromodesoxiuridina/metabolismo , Células COS , Ciclo Celular/genética , Quinasa de Punto de Control 2 , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , Embrión no Mamífero/embriología , Factores de Unión al ADN Específico de las Células Eritroides/deficiencia , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Heterocromatina/metabolismo , Inmunohistoquímica , Hibridación in Situ , Microscopía Fluorescente , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/metabolismoRESUMEN
MicroRNAs (miRNAs) control tissue development, but their mechanism of regulation is not well understood. We used a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. Two conserved miRNAs, miR 144 and miR 451, emerged as direct targets of the critical hematopoietic transcription factor GATA-1. In vivo, GATA-1 binds a distal upstream regulatory element to activate RNA polymerase II-mediated transcription of a single common precursor RNA (pri-miRNA) encoding both mature miRNAs. Zebrafish embryos depleted of miR 451 by using antisense morpholinos form erythroid precursors, but their development into mature circulating red blood cells is strongly and specifically impaired. These results reveal a miRNA locus that is required for erythropoiesis and uncover a new regulatory axis through which GATA-1 controls this process.
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Células Precursoras Eritroides/citología , Eritropoyesis/genética , Factor de Transcripción GATA1/fisiología , MicroARNs/fisiología , Animales , Línea Celular Tumoral , Factores de Unión al ADN Específico de las Células Eritroides , Hibridación in Situ , Ratones , MicroARNs/análisis , Análisis por Micromatrices , Pez CebraRESUMEN
OBJECTIVE: In search of functional polymorphisms associated with the genetics of insulin resistance, we studied a variant in the promoter of PIK3CB, the gene coding for the catalytic p110beta subunit of phosphatidylinositol (PI) 3-kinase, a major effector of insulin action. RESEARCH DESIGN AND METHODS: The rs361072 C/T variant was selected among single nucleotide polymorphisms of the PIK3CB region because we suspected that its common C allele (allelic frequency approximately 50% in Europeans) could create a GATA-binding motif and was genotyped in five cohorts of obese (n = 1,876) and two cohorts of nonobese (n = 1,490) European children. To estimate insulin resistance in these children, the homeostasis model assessment for insulin resistance (HOMA-IR) index was measured in strict nutritional conditions. GATA-binding and functional effects of rs361072 were explored in transfected cell lines and in lymphocytes from obese children. RESULTS: The rs361072 C/T variant was associated with HOMA-IR in the obese children cohorts (1.7 x 10(-12) < P < 2 x 10(-4) for C/C vs. T/T using regression analysis). HOMA-IR averaged 3.3 +/- 0.1 in C/C and 4.5 +/- 0.2 in T/T obese children (P = 4.5 x 10(-6) by ANOVA). C/T patients had intermediate values. As shown by the interaction between BMI and genotype (P = 2.1 x 10(-9)), the association of rs361072 with HOMA-IR depended on BMI and was only marginal in nonobese children (P = 0.04). At the molecular level, the C allele of rs361072 was found to create a GATA-binding site able to increase transcription of PIK3CB. CONCLUSIONS: We postulate that the C allele of rs361072 is a causal variant capable of attenuating insulin resistance in obese children through increased expression of p110beta.
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Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Variación Genética , Resistencia a la Insulina/genética , Obesidad/genética , Fosfatidilinositol 3-Quinasas/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Sitios de Unión , Glucemia/análisis , Índice de Masa Corporal , Niño , Fosfatidilinositol 3-Quinasas Clase II , Estudios de Cohortes , Predisposición Genética a la Enfermedad , Genotipo , Homeostasis , Humanos , Insulina/sangre , Regiones Promotoras Genéticas , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: To characterize how interleukin-3 and erythropoietin regulate cell fate by modulating the expression of lineage-specific transcription factors. METHODS: This study analyzed mRNA and protein levels, gene transcription rates, and mRNA and protein stabilities of erythroid-specific transcription factors in lineage-restricted cells derived from the 32D cell line cultured either in interleukin-3 or erythropoietin. RESULTS: Erythroid 32D subclones expressed levels of Idl, Gata-2, and Scl comparable and levels of Eklf and Gata-1 higher than those expressed by myeloid subclones. While maintained in interleukin-3, erythroid cells remained immature despite their high expression of Gata-1, Gata-2, Scl, Eklf, and Idl. Switching the erythroid cells to erythropoietin induced cell maturation (within 48 hours) and reduced expression of Gata-2 and Idl (in 24 hours) but did not alter the expression of Gata-1. The effects of interleukin-3 were mostly mediated by increases in transcription rates (Scl and Gata-2), and that of erythropoietin was apparently due to increased mRNA and protein (Gata-1, Scl, and Eklf) stability. In particular, erythropoietin increased the stability of the processed and transcriptionally more active form of GATA-1 protein. CONCLUSIONS: These results suggest that interleukin-3 and erythropoietin cooperate to establish the lineage-specific transcription factor milieu of erythroid cells: interleukin-3 regulates mainly gene transcription and erythropoietin consistently increases mRNA and protein stability.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Eritroides/efectos de los fármacos , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Eritropoyetina/fisiología , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Interleucina-3/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Células Eritroides/citología , Factores de Unión al ADN Específico de las Células Eritroides/efectos de los fármacos , Factores de Unión al ADN Específico de las Células Eritroides/genética , Eritropoyetina/farmacología , Perfilación de la Expresión Génica , Proteína 1 Inhibidora de la Diferenciación/efectos de los fármacos , Proteína 1 Inhibidora de la Diferenciación/genética , Interleucina-3/farmacología , Ratones , Fenotipo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de TiempoRESUMEN
Hematopoiesis is a complex process which gives rise to all blood lineages in the course of an organism's lifespan. However, the underlying molecular mechanism governing this process is not fully understood. Here we report the isolation and detailed study of a newly identified zebrafish ugly duckling (Udu) mutant allele, Udu(sq1). We show that loss-of-function mutation in the udu gene disrupts primitive erythroid cell proliferation and differentiation in a cell-autonomous manner, resulting in red blood cell (RBC) hypoplasia. Positional cloning reveals that the Udu gene encodes a novel factor that contains 2 paired amphipathic alpha-helix-like (PAH-L) repeats and a putative SANT-L (SW13, ADA2, N-Cor, and TFIIIB-like) domain. We further show that the Udu protein is predominantly localized in the nucleus and deletion of the putative SANT-L domain abolishes its function. Our study indicates that the Udu protein is very likely to function as a transcription modulator essential for the proliferation and differentiation of erythroid lineage.
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Células Precursoras Eritroides/citología , Factores de Unión al ADN Específico de las Células Eritroides/fisiología , Eritropoyesis , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Eritrocitos Anormales/patología , Células Eritroides/citología , Factores de Unión al ADN Específico de las Células Eritroides/genética , Mutación , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
In an adenosine triphosphate (ATP)-dependent process, the hSWI/SNF chromatin remodeling complex functions to alter chromatin structure, thereby regulating transcription factor access to DNA. In addition to interactions with transcription factors and recognition of acetylated histone residues, the chromatin remodeling activity of hSWI/SNF has also been shown to respond to a variety of cell signaling pathways. Our results demonstrate a novel interaction between the serine/threonine kinase Akt and members of the hSWI/SNF chromatin remodeling complex. Activation of Akt in HeLa cells resulted in its association with hSWI/SNF subunits: INI1, BAF155 and BAF170, as well as actin. BAF155 became preferentially recognized by an antibody that detects phosphorylated Akt substrates upon activation of Akt, suggesting that BAF155 may be an in vivo target for phosphorylation by Akt. Glutathione-S-transferase (GST) pulldown experiments demonstrated that INI1 and BAF155 were both capable of directly interacting with Akt. Finally, in vitro kinase assays provided additional evidence that BAF155 and potentially INI1 are substrates for Akt phosphorylation. These data provide the first evidence that Akt signaling may modulate function of the hSWI/SNF complex.
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Cromatina/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Células HeLa , Humanos , Fosforilación , Unión Proteica , Proteína SMARCB1 , Factores de Transcripción/metabolismoRESUMEN
CCAAT/enhancer-binding protein beta (C/EBPbeta) can function as a repressor or as an activator of human papillomavirus (HPV) gene expression, depending on which cell type the experiments are conducted. In this report, it was shown that within primary human foreskin keratinocyte cells (HFK) the activity of C/EBPbeta can be switched from that of a repressor of HPV11 expression to an activator by mutating a single promoter-proximal consensus YY1-binding site within the HPV11 upstream regulatory region (URR). It was shown that in HFK cells, exogenous expression of C/EBPbeta significantly activates the expression of mutant HPV11 URR reporter plasmids that contain deletions which overlap a 127 bp region (-269 to -142). Inclusive in this region are binding sites for multiple transcription factors, including AP1, YY1 and C/EBPalpha. Only mutation of the YY1 site resulted in the switch in phenotype, indicating that C/EBPbeta represses HPV11 expression in these cells via YY1 binding. The level of YY1 activity was also measured in HFK cells transfected with a C/EBPbeta expression plasmid and a significant increase in YY1 activity as compared with mock-transfected cells was found. C33A cells, which exhibit activation of wild-type HPV11 gene expression with exogenous C/EBPbeta co-expression, failed to demonstrate C/EBPbeta-induced YY1 activation. It was concluded that in HFK cells, exogenous C/EBPbeta induces the activity of YY1, which, in turn, can repress HPV11 URR expression through the promoter-proximal YY1-binding site.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Papillomaviridae/metabolismo , Regiones Promotoras Genéticas/fisiología , Factor de Transcripción YY1/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/biosíntesis , Células Cultivadas , Humanos , Queratinocitos/metabolismo , Papillomaviridae/genética , Proteínas Represoras/biosíntesis , PielRESUMEN
Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.
Asunto(s)
Factores de Transcripción Fushi Tarazu/genética , Procesos de Determinación del Sexo , Diferenciación Sexual/fisiología , Pez Cebra/genética , Animales , Hormona Antimülleriana , Aromatasa/genética , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides/genética , Femenino , Glicoproteínas/genética , Proteínas HMGB/genética , Proteínas de Homeodominio/genética , Masculino , Modelos Genéticos , Receptores Citoplasmáticos y Nucleares/genética , Factor de Transcripción SOX9 , Factor Esteroidogénico 1 , Hormonas Testiculares/genética , Factores de Transcripción/genética , Proteínas WT1/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The primary genetic lesion(s), as well as the biological processes responsible for the typical structural changes of the bone marrow microenvironment in idiopathic myelofibrosis, are still poorly understood, although a central role in disease pathogenesis has been attributed to the clonal proliferation and defective maturation of megakaryocytes. Two animal models of the disease have been described, that in the last few years significantly contributed to the elucidation of some of the pathogenetic steps of the human disease; these are represented by mice genetically modified to overexpress thrombopoietin and by knock-down mice with defective GATA-1 expression in megakaryocytes (GATA-1(low) mice). This review will outline these murine models, both characterized by extensive accumulation of megakaryocytes in hematopoietic tissues, and illustrate how they provided insights into the identification of some of the molecules and mechanisms responsible for the development of fibrosis and osteosclerosis that present major similarities with those observed in patients with idiopathic myelofibrosis.
Asunto(s)
Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Ratones Noqueados , Ratones Transgénicos , Mielofibrosis Primaria/fisiopatología , Trombopoyetina , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Técnicas de Transferencia de Gen , Ratones , Mielofibrosis Primaria/inmunología , Proteínas/metabolismo , Trombopoyetina/genética , Trombopoyetina/inmunología , Trombopoyetina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
The stem cell leukemia (SCL) transcription factor is essential for vertebrate hematopoiesis. Using the powerful zebrafish model for embryonic analysis, we compared the effects of either reducing or ablating Scl using morpholino-modified antisense RNAs. Ablation of Scl resulted in the loss of primitive and definitive hematopoiesis, consistent with its essential role in these processes. Interestingly, in embryos with severely reduced Scl levels, erythroid progenitors expressing gata1 and embryonic globin developed. Erythroid maturation was deficient in these Scl hypomorphs, supporting that Scl was required both for the erythroid specification and for the maturation steps, with maturation requiring higher Scl levels than specification. Although all hematopoietic functions were rescued by wild-type Scl mRNA, an Scl DNA binding mutant rescued primitive and definitive hematopoiesis but did not rescue primitive erythroid maturation. Together, we showed that there is a distinct Scl hypomorphic phenotype and demonstrated that distinct functions are required for the roles of Scl in the specification and differentiation of primitive and definitive hematopoietic lineages. Our results revealed that Scl participates in multiple processes requiring different levels and functions. Further, we identified an Scl hypomorphic phenotype distinct from the null state.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas/fisiología , Proteínas de Pez Cebra/fisiología , Empalme Alternativo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Linaje de la Célula , ADN/química , Proteínas de Unión al ADN , Electroforesis en Gel de Agar , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Hematopoyesis , Hibridación in Situ , Modelos Genéticos , Mutación , Fenotipo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , ARN/química , ARN sin Sentido/química , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. Our laboratory has previously defined the human Gfi-1B promoter region and shown that GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, here we report that (i) Gfi-1B transcription is negatively regulated by its own gene product, (ii) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (iii) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, we propose that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells.