Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression.     

Distribution of cholinergic neuronal differentiation factor/leukemia inhibitory factor binding sites in the developing and adult rat nervous system in vivo

Cholinergic neuronal differentiation factor/leukemia inhibitory factor (CDF/LIF) is a multi-functional cytokine that affects neurons as well as many other cell types. Toward elucidating its neural functions in vivo, we previously investigated the distribution of CDF/LIF binding sites with iodinated native CDF/LIF in embryonic to postnatal day 0 (P0) rats. In the present study, we have extended our examination to postnatal ages and find that specific CDF/LIF binding sites are present at defined developmental stages in additional brain regions not previously exhibiting binding by P0. High levels of binding are detected in all P7 sensory and autonomic ganglia examined, but only in restricted postnatal central nervous system structures. Cranial motor and mesencephalic trigeminal neurons maintain high levels throughout, while binding to spinal motor neurons, which decreases to low levels at P0, reappears by P14 and increases with age. Most other structures, which show detectable binding by P0, exhibit higher levels at postnatal ages, including the red, deep, ventral cochlear, trapezoid, superior olivary, vestibular, ventral tegmental, and ventral posterior thalamic nuclei as well as the glomerular layer of the olfactory bulb. High levels are also detected in several structures for the first time after P0, including the cerebellar cortex (molecular and Purkinje cell layers), lateral reticular nucleus of the medulla and reticular formation, as well as the reticulotegmental, medial geniculate, solitary (rostral, dorsomedial, and commissural regions), medial septal, lateral mammillary, and lateral habenular nuclei. These results not only identify regions of potential CDF/LIF-responsive neurons and glia throughout development but suggest new CDF/LIF roles in the nervous system. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 163–192, 1997.     

神经元限制性沉默因子在NTera2/CloneD1细胞向神经元分化模型中表达的检测

目的:建立NTera2/CloneD1细胞向神经元分化的模型,检测神经元限制性沉默因子(NRSF)经分化培养基诱导后表达的变化。方法:收集正常培养的NTera2/CloneD1细胞及经全反式维甲酸(RA)、阿糖胞苷(AraC)、尿苷分阶段诱导共28 d的细胞,显微镜下观察诱导前后细胞的形态学变化;免疫荧光法检测NTera2/CloneD1细胞诱导前后干性标志Nestin、Sox2和成熟神经元特异性标志NF-200、β-tubulinⅢ的表达情况;应用RT-PCR和免疫荧光法对NRSF进行mRNA和蛋白水平的检测。结果:显微镜下观察到正常培养的NTera2/CloneD1细胞呈克隆样生长,经分化培养基诱导后的NTera2/CloneD1细胞表现出典型的神经元样细胞形态。免疫荧光检测表明,未诱导的NTera2/CloneD1细胞表达神经干细胞的标志Sox2、Nestin,不表达成熟神经元特异性蛋白NF-200、β-tubulinⅢ;而经RA等诱导分化的细胞则不表达Sox2、Nestin,表达NF-200、β-tubulinⅢ。RT-PCR和免疫荧光检测显示,NRSF在诱导分化后的NTera2/CloneD1细胞中的表达量显著降低。结论:建立了NTera2/CloneD1细胞向神经元分化的模型,NRSF在诱导后的NTera2/CloneD1细胞中表达量显著下调,提示NTera2/CloneD1细胞在诱导过程中可能通过下调NRSF,使受到NRSF负性调控的神经元特异性蛋白启动表达并上调,进而实现NTera2/CloneD1细胞向神经元的定向分化。