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Granule neuron precursors (GNPs) are the most actively proliferating cells in the postnatal nervous system, and mutations in pathways that control the GNP cell cycle can result in medulloblastoma. The transcription factor Atoh1 has been suspected to contribute to GNP proliferation, but its role in normal and neoplastic postnatal cerebellar development remains unexplored. We show that Atoh1 regulates the signal transduction pathway of Sonic Hedgehog, an extracellular factor that is essential for GNP proliferation, and demonstrate that deletion of Atoh1 prevents cerebellar neoplasia in a mouse model of medulloblastoma. Our data shed light on the function of Atoh1 in postnatal cerebellar development and identify a new mechanism that can be targeted to regulate medulloblastoma formation. [ABSTRACT FROM AUTHOR]

Proneural factors represent <10 transcriptional regulators required for specifying all of the different neurons of the mammalian nervous system. The mechanisms by which such a small number of factors creates this diversity are still unknown. We propose that proteins interacting with proneural factors confer such specificity. To test this hypothesis we isolated proteins that interact with Math1, a proneural transcription factor essential for the establishment of a neural progenitor population (rhombic lip) that gives rise to multiple hindbrain structures and identified the E-protein Tcf4. Interestingly, haploin-sufficiency of TCF4 causes the Pitt—Hopkins mental retardation syndrome, underscoring the important role for this protein in neural development. To investigate the functional relevance of the Math1/Tcf4 interaction in vivo, we studied Tcf4-/- mice and found that they have disrupted pontine nucleus development. Surprisingly, this selective deficit occurs without affecting other rhombic lip-derived nuclei, despite expression of Math1 and Tcf4 throughout the rhombic lip. Importantly, deletion of any of the other E-protein-encoding genes does not have detectable effects on Math1-dependent neurons, suggesting a specialized role for Tcf4 in distinct neural progenitors. Our findings provide the first in vivo evidence for an exclusive function of dimers formed between a proneural basic helix—loop—helix factor and a specific E-protein, offering insight about the mechanisms underlying transcriptional programs that regulate development of the mammalian nervous system. [ABSTRACT FROM AUTHOR]

PHOX2A is a paired-like homeodomain transcription factor that participates in specifying the autonomic nervous system. It is also involved in the transcriptional control of the noradrenergic neurotransmitter phenotype as it regulates the gene expression of tyrosine hydroxylase and dopamine-β-hydroxylase. The results of this study show that the human orthologue of PHOX2A is also capable of regulating the transcription of the human α3 nicotinic acetylcholine receptor gene, which encodes the ligand-binding subunit of the ganglionic type nicotinic receptor. In particular, we demonstrated by chromatin immunoprecipitation and DNA pulldown assays that PHOX2A assembles on the SacI-NcoI region of α3 promoter and, by co-transfection experiments, that it exerts its transcriptional effects by acting through the 60-bp minimal promoter. PHOX2A does not seem to bind to DNA directly, and its DNA binding domain seems to be partially dispensable for the regulation of α3 gene transcription. However, as suggested by the findings of our co-immunoprecipitation assays, it may establish direct or indirect protein-protein interactions with Sp1, thus regulating the expression of α3 through a DNA-independent mechanism. As the α3 subunit is expressed in every terminally differentiated ganglionic cell, this is the first example of a "pan-autonomic" gene whose expression is regulated by PHOX2 proteins. [ABSTRACT FROM AUTHOR]

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomaln transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5′-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription. [ABSTRACT FROM AUTHOR]

Abstract: The dicarboximide fungicide iprodione (Ip) causes oxidative damage as a result of the production of free oxygen radicals, and induces cytochrome P4501A3 (CYP1A3) in cultured rainbow trout hepatocytes. The aim of this study was to characterise some of the molecular mechanisms by means of which Ip activates the aryl hydrocarbon receptor (AhR) and subsequently induces the CYP1A3 gene in rainbow trout (Oncorhynchus mykiss). The study was performed using primary hepatocytes and transfected HepG2 cells with a reporter construct, in which luciferase gene expression is under the transcriptional control of a multimerised xenobiotic response elements (4XREs), or a 2.3Kb DNA fragment (corresponding to the trout CYP1A3 gene promoter). Ip exposure increased rainbow trout hepatocyte CYP1A3 mRNA over time and increased the expression of reporter gene in HepG2, thus suggesting that Ip induces the CYP1A3 gene by activating the AhR. Genistein, a tyrosine kinase inhibitor, efficiently inhibited the Ip-mediated induction of the CYP1A3 gene as demonstrated by mRNA level decrease and the impaired activation of the luciferase reporter gene constructs. Staurosporine, an inhibitor of protein kinase C, also suppressed the induction by Ip. When the AhR antagonist α-naphthoflavone was added to the cultures, Ip-mediated CYP1A3 induction was suppressed. These findings are consistent with a mechanism of Ip-mediated CYP1A3 gene induction that involves the activation of the AhR complex via phosphorylation–dephosphorylation reactions. [Copyright &y& Elsevier]

Abstract: The mRNA encoding the human α5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the α5 transcript was also detected in several non-neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed α5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of α5 in neuronal and nonneuronal cells, we isolated the 5′-regulatory region of the human gene and characterized its properties. Here we identify the α5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue-specific fashion. In particular, the segment specifying the 5′-untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position- and orientation-independent fashion. We therefore conclude that the expression of α5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs. [ABSTRACT FROM AUTHOR]

The proneural, basic helix-loop-helix transcription factor Atoh1 governs the development of numerous key neuronal subtypes, such as cerebellar granule and brainstem neurons, inner ear hair cells, and several neurons of the proprioceptive system, as well as diverse nonneuronal cell types, such as Merkel cells and intestinal secretory lineages. However, the mere handful of targets that have been identified barely begin to account for Atoh1's astonishing range of functions, which also encompasses seemingly paradoxical activities, such as promoting cell proliferation and medulloblastoma formation in the cerebellum and inducing cell cycle exit and suppressing tumorigenesis in the intestine. We used a multipronged approach to create a comprehensive, unbiased list of over 600 direct Atoh1 target genes in the postnatal cerebellum. We found that Atoh1 binds to a 10 nucleotide motif (AtEAM) to directly regulate genes involved in migration, cell adhesion, metabolism, and other previously unsuspected functions. This study expands current thinking about the transcriptional activities driving neuronal differentiation and provides a framework for further neurodevelopmental studies. [ABSTRACT FROM AUTHOR]

C-myb and B-myb belong to the myb family of transcription factors. We have shown previously that c-myb is deregulated in fibroblasts from systemic sclerosis (scleroderma) patients relative to normal fibroblasts. Scleroderma fibroblasts are known to express elevated levels of collagen genes and transforming growth factor β is known to be a pro-fibrotic cytokine and to induce transcription of type I collagen genes. We have therefore investigated the role of c-myb and B-myb in the regulation of type I collagen genes in response to transforming growth factor β in normal human fibroblasts. We show that, in these cells, transforming growth factor β treatment induces c-myb as well as collagen α1(I) and α2(I) gene expression, but not B-myb. Furthermore we demonstrate by co-transfection assays that c-myb can upregulate α1(I) and α2(I) collagen promoters by 6–10-fold whereas B-myb is inactive. The activity of c-myb on both type I collagen promoters requires a functional c-myb DNA binding domain suggesting a direct interaction between c-myb and these promoters. Indeed c-myb is active also on a 500 bp fragment of the α2(I) collagen promoter and can bind to this fragment in electrophoretic mobility shift assays. Finally, we show that anti-c-myb anti-sense treatment reduces α1(I) and to a lesser extent α2(I) collagen gene expression. These data strongly suggest that c-myb, but not B-myb, plays a direct role in the upregulation of type I collagen gene expression in response to transforming growth factor β. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). To determine the long-term effects of exercise, we implemented a mild exercise regimen in a mouse model of SCA1 and found a considerable improvement in survival accompanied by up-regulation of epidermal growth factor and consequential down-regulation of Capicua, which is an ATXN1 interactor. Offspring of Capicua mutant mice bred to SCA1 mice showed significant improvement of air disease phenotypes. Although polyglutamine-expanded Atxn1 caused some loss of Capicua function, further reduction of Capicua levels—either genetically or by exercise--mitigated the disease phenotypes by dampening the toxic gain of function. Thus, exercise might have long-term beneficial effects in other ataxias and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Summary: Mice lacking the proneural transcription factor Math1 (Atoh1) lack multiple neurons of the proprioceptive and arousal systems and die shortly after birth from an apparent inability to initiate respiration. We sought to determine whether Math1 was necessary for the development of hindbrain nuclei involved in respiratory rhythm generation, such as the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN), defects in which are associated with congenital central hypoventilation syndrome (CCHS). We generated a Math1-GFP fusion allele to trace the development of Math1-expressing pFRG/RTN and paratrigeminal neurons and found that loss of Math1 did indeed disrupt their migration and differentiation. We also identified Math1-dependent neurons and their projections near the pre-Bötzinger complex, a structure critical for respiratory rhythmogenesis, and found that glutamatergic modulation reestablished a rhythm in the absence of Math1. This study identifies Math1-dependent neurons that are critical for perinatal breathing that may link proprioception and arousal with respiration. [Copyright &y& Elsevier]

Mammalian NADH-cytochrome b(5) reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R. [ABSTRACT FROM AUTHOR]