RESUMEN
Heterozygous GATA2 mutations underlie an array of complex hematopoietic and lymphatic diseases. Analysis of the literature reporting three recurrent GATA2 germline (g) mutations (gT354M, gR396Q and gR398W) revealed different phenotype tendencies. Although all three mutants differentially predispose to myeloid malignancies, there was no difference in leukemia-free survival for GATA2 patients. Despite intense interest, the molecular pathogenesis of GATA2 mutation is poorly understood. We functionally characterized a GATA2 mutant allelic series representing major disease phenotypes caused by germline and somatic (s) mutations in zinc finger 2 (ZF2). All GATA2 mutants, except for sL359V, displayed reduced DNA-binding affinity and transactivation compared with wild type (WT), which could be attributed to mutations of arginines critical for DNA binding or amino acids required for ZF2 domain structural integrity. Two GATA2 mutants (gT354M and gC373R) bound the key hematopoietic differentiation factor PU.1 more strongly than WT potentially perturbing differentiation via sequestration of PU.1. Unlike WT, all mutants failed to suppress colony formation and some mutants skewed cell fate to granulocytes, consistent with the monocytopenia phenotype seen in GATA2-related immunodeficiency disorders. These findings implicate perturbations of GATA2 function shaping the course of development of myeloid malignancy subtypes and strengthen complete or nearly complete haploinsufficiency for predisposition to lymphedema.
Asunto(s)
Diferenciación Celular/genética , Factor de Transcripción GATA2/genética , Sistema Hematopoyético/patología , Mutación/genética , Transcripción Genética/genética , Animales , Células COS , Chlorocebus aethiops , Femenino , Predisposición Genética a la Enfermedad/genética , Genotipo , Células HEK293 , Haploinsuficiencia/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoAsunto(s)
Factor de Transcripción GATA2/genética , Mutación de Línea Germinal/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Elementos de Respuesta/genética , Trombocitopenia/genética , Western Blotting , Femenino , Células HEK293 , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patología , Linaje , Trombocitopenia/patologíaRESUMEN
Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.
Asunto(s)
Perfilación de la Expresión Génica , Neovascularización Fisiológica/genética , Transducción de Señal , Teorema de Bayes , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Factor Inhibidor de Leucemia/genética , Sistema de Señalización de MAP Quinasas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo , Transcripción Genética/genéticaRESUMEN
Catalytically active biotin protein ligase from Saccharomyces cerevisiae (EC 6.3.4.15) was overexpressed in Escherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. coli strains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.
