RESUMO
Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl(-/-) embryos. However, here we demonstrate by using a knockout approach that, within the endogenous scl locus, the +18/19 enhancer is not necessary for the initiation of scl transcription or for the formation of hematopoietic cells. These results led to the identification of a bifunctional 5' enhancer (-3.8 element), which targets expression to hematopoietic progenitors and endothelium, contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. These data demonstrate that two geographically distinct but functionally related enhancers regulate scl transcription in hematopoietic progenitors and endothelial cells and suggest that enhancers with dual hematopoietic-endothelial activity may represent a general strategy for regulating blood and endothelial development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares , Proteína Proto-Oncogênica c-fli-1 , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Homologous recombination in ES cells was employed to generate mice with targeted deletion of the first three exons of the gamma-synuclein gene. Complete inactivation of gene expression in null mutant mice was confirmed on the mRNA and protein levels. Null mutant mice are viable, are fertile, and do not display evident phenotypical abnormalities. The effects of gamma-synuclein deficiency on motor and peripheral sensory neurons were studied by various methods in vivo and in vitro. These two types of neurons were selected because they both express high levels of gamma-synuclein from the early stages of mouse embryonic development but later in the development they display different patterns of intracellular compartmentalization of the protein. We found no difference in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion. The survival of gamma-synuclein-deficient trigeminal neurons in various culture conditions was not different from that of wild-type neurons. There was no difference in the numbers of myelinated and nonmyelinated fibers in the saphenous nerves of these animals, and sensory reflex thresholds were also intact in gamma-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Taken together, our data suggest that like alpha-synuclein, gamma-synuclein is dispensable for the development and function of the nervous system.