Dataset Information

Septamer element-binding proteins in neuronal and glial differentiation.

ABSTRACT: Differentiation of progenitors into neurons and glia is regulated by interactions between regulatory DNA elements of neuron- and glia-specific genes and transcription factors that are differentially expressed by progenitors at progressive stages of neural development. We have identified a novel DNA regulatory element (TTTGCAT = septamer) present on the enkephalin (ENK), neuronal cell adhesion molecule, neurofilament of 68 kDa (NF68), growth-associated protein of 43 kDa, glial high-affinity glutamine transporter, tyrosine hydroxylase, etc., genes. When septamer function was blocked by introducing septamer competitor DNA into primary differentiating neural cultures, mRNA levels of ENK, NF68, and glial fibrillary acidic protein decreased by 50-80%, whereas no effect was seen using a control DNA. Septamer elements serve as binding sites for lineage-specific multimeric complexes assembled from three distinct nuclear proteins. Progenitors express a 16 kDa protein (p-sept) which binds to DNA as a homodimer (detected as the 32 kDa P-band). Cells that entered the neuronal lineage express an additional 29 kDa protein (n-sept) that binds to the homodimerized p-sept, and together they form a 62 kDa multimer (detected as N-band). Cells that entered the glial lineage express a distinct 23 kDa protein (g-sept), which along with the homodimerized p-sept form a 56 kDa multimer (observed as G-band). The binding of the distinct protein complexes (P, G, and N) to the septamer site causes a lineage-specific DNA bending (P = 53 degrees; G = 72 degrees; and N = 90 degrees ), which may contribute to the regulatory effect of the septamer interaction. In summary, septamer and its binding proteins represent novel protein-DNA interactions that may contribute to the regulation of neuroglial differentiation in the developing mammalian CNS.

SUBMITTER: Dobi A

PROVIDER: S-EPMC6774153 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Septamer element-binding proteins in neuronal and glial differentiation.

Dobi A A Palkovits M M Palkovits C G CG Ring M A MA Agoston D D

The Journal of neuroscience : the official journal of the Society for Neuroscience 20000201 3

Differentiation of progenitors into neurons and glia is regulated by interactions between regulatory DNA elements of neuron- and glia-specific genes and transcription factors that are differentially expressed by progenitors at progressive stages of neural development. We have identified a novel DNA regulatory element (TTTGCAT = septamer) present on the enkephalin (ENK), neuronal cell adhesion molecule, neurofilament of 68 kDa (NF68), growth-associated protein of 43 kDa, glial high-affinity gluta ...[more]

PMID: 10648713

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Project description:The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial version of the "Color Timer" mouse system will provide a powerful new tool for neurogenesis research.

Project description:BackgroundNotch signaling has been conserved throughout evolution and plays a fundamental role in various neural developmental processes and the pathogenesis of several human cancers and genetic disorders. However, how Notch signaling regulates various cellular processes remains unclear. Although Deltex proteins have been identified as cytoplasmic downstream elements of the Notch signaling pathway, few studies have been reported on their physiological role.ResultsWe isolated zebrafish deltex1 (dtx1) and showed that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. The transcription of dtx1 was suppressed by the direct binding of the Notch downstream transcription factors Her2 and Her8a. Overexpressing the complete coding sequence of Dtx1 was necessary for inducing neuronal and glial differentiation. By contrast, disrupting Dtx1 expression by using a Dtx1 construct without the RING finger domain reduced neuronal and glial differentiation. This effect was phenocopied by the knockdown of endogenous Dtx1 expression by using morpholinos, demonstrating the essential function of the RING finger domain and confirming the knockdown specificity. Cell proliferation and apoptosis were unaltered in Dtx1-overexpressed and -deficient zebrafish embryos. Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway. However, both Dtx1 and Notch activation induced glial differentiation, and Dtx1 and Notch activation negatively inhibited each other in a reciprocal manner, which achieves a proper balance for the expression of Dtx1 and Notch to facilitate glial differentiation. We further confirmed that the Dtx1-Notch-Hairy/E(Spl) cascade was sufficient to induce neuronal and glial differentiation by concomitant injection of an active form of Notch with dtx1, which rescued the neuronogenic and gliogenic defects caused by the activation of Notch signaling.ConclusionsOur results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation. Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.

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Septamer element-binding proteins in neuronal and glial differentiation.

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Septamer element-binding proteins in neuronal and glial differentiation.

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