The circadian clock controls many physiological parameters including immune response to infectious agents, which is mediated by activation of the transcription factor NF-κB. It is widely accepted that circadian regulation is based on periodic changes in gene expression that are triggered by transcriptional activity of the CLOCK/BMAL1 complex. Through the use of a mouse model system we show that daily variations in the intensity of the NF-κB response to a variety of immunomodulators are mediated by core circadian protein CLOCK, which can up-regulate NF-κB-mediated transcription in the absence of BMAL1; moreover, BMAL1 counteracts the CLOCK-dependent increase in the activation of NF-κB-responsive genes. Consistent with its regulatory function, CLOCK is found in protein complexes with the p65 subunit of NF-κB, and its overexpression correlates with an increase in specific phosphorylated and acetylated transcriptionally active forms of p65. In addition, activation of NF-κB in response to immunostimuli in mouse embryonic fibroblasts and primary hepatocytes isolated from Clock-deficient mice is significantly reduced compared with WT cells, whereas Clock-Δ19 mutation, which reduces the transactivation capacity of CLOCK on E-box-containing circadian promoters, has no effect on the ability of CLOCK to up-regulate NF-κB-responsive promoters. These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively.

The mammalian POU-domain factor Brn-3.0 (Brn-3, Brn-3a) is a member of the POU-IV class of transcription factors which resemble the C. elegans factor unc-86 in structure, DNA-binding properties and expression in subsets of sensory neurons. Using specific antisera, we have explored the expression of Brn-3.0 in the early development of the mouse nervous system. Brn-3.0 expression begins at embryonic day 8.5 (E8.5) in a specific set of midbrain tectal neurons whose time and place of appearance are consistent with the earliest CNS neurons previously identified using non-specific markers of neural differentiation. By E9.5, Brn-3.0 immunoreactivity also identifies early CNS neurons in the hindbrain and spinal cord. In the peripheral sensory ganglia, Brn-3.0 expression is first observed at E9.0 in migrating precursors of the trigeminal ganglion, followed by the other sensory cranial and dorsal root ganglia, in a rostral to caudal sequence. Double-label immunofluorescence with Brn-3.0 and the markers of cell division PCNA and BrdU demonstrate that Brn-3.0 is restricted to the post-mitotic phase of CNS development. In the sensory cranial and dorsal root ganglia, however, Brn-3.0 is expressed in dividing neural precursors, suggesting that the nature or timing of developmental events controlled by Brn-3.0 are distinct in the CNS and peripheral neurons. Restriction of Brn-3.0 expression to post-mitotic CNS neurons demonstrates that Brn-3.0 is not required for neurogenesis or patterning of the neuroepithelium in the CNS, but suggests a role in specification of mature neuronal phenotypes.

Brn3a/Brn-3.0 is a POU-domain transcription factor expressed in primary sensory neurons of the cranial and dorsal root ganglia and in specific neurons in the caudal CNS. Mice lacking Brn3a undergo extensive sensory neural death late in gestation and die at birth. To further examine Brn3a expression and the abnormalities that accompany its absence, we constructed a transgene containing 11 kb of Brn3a upstream regulatory sequence linked to a LacZ reporter. Here we show that these regulatory sequences direct transgene expression specifically to Brn3a peripheral sensory neurons of the cranial and dorsal root ganglia. Furthermore, expression of the 11 kb/LacZ reporter in the sensory neurons of the mesencephalic trigeminal, but not other Brn3a midbrain neurons, demonstrates that cellspecific transgene expression is targeted to a functional class of neurons rather than to an anatomical region. We then interbred the 11 kb/LacZ reporter strain with mice carrying a null mutant allele of Brn3a to generate 11 kb/LacZ, Brn3a knockout mice. ␤-Galactosidase expression in these mice reveals significant axonal growth defects, including excessive and premature branching of the major divisions of the trigeminal nerve and a failure to correctly innervate whisker follicles, all of which precede sensory neural death in these mice. These defects in Brn3a Ϫ/Ϫ mice resemble strongly those seen in mice lacking the mediators of sensory pathfinding semaphorin 3A and neuropilin-1. Here we show, however, that sensory neurons are able to express neuropilin-1 in the absence of Brn3a. Key words: POU-domain; homeodomain; Brn3; TrkC; trigeminal ganglion; sensory ganglion; axon guidanceThe vertebrate nervous system contains a large number of specific cell types, and how this neuronal diversity is generated is a central question in the study of brain development. Many neurons in the developing brain can be recognized by the expression of specific "neural identity" genes that are first detectable at approximately the time of exit from cell division and may persist throughout embryogenesis. Most such genes discovered to date have been transcription factors, often but not always containing a homeodomain motif (Rubenstein and Puelles, 1994). A specific neural identity gene may be expressed in a recognizable class of neurons, such as the motor neurons of the brainstem and spinal cord, or it may characterize a group of cells that have no other known features in common. Accordingly, they are good candidates for establishing the neuronal phenotypes characterized by, among others, the cellspecific expression of neurotransmitters and their receptors, axonal guidance to selected targets, and synaptic specificity. However, the neuronal properties that are actually regulated by these factors, and how this regulation is accomplished, remain primarily undiscovered.The "Brn3" or POU-IV class of transcription factors is comprised of three members in vertebrates that share very similar DNA recognition properties to their invertebrate counterparts (Gruber et al., 199...

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor.

SUMMARY Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was down-regulated in invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR down-regulates the amounts of several GTP-bound (active) RHO-GTPases, suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix and invade in vitro and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed, whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identified GMPR as a melanoma invasion suppressor, and established a link between guanosine metabolism and RHO-GTPase-dependent melanoma cell invasion.

Brn3a is a POU-domain transcription factor expressed in peripheral sensory neurons and in specific interneurons of the caudal CNS. Sensory expression of Brn3a is regulated by a specific upstream enhancer, the activity of which is greatly increased in Brn3a knockout mice, implying that Brn3a negatively regulates its own expression. Brn3a binds to highly conserved sites within this enhancer, and alteration of these sites abolishes Brn3a regulation of reporter transgenes. Furthermore, endogenous Brn3a expression levels in the sensory ganglia of Brn3a +/+ and Brn3a +/-mice are similar, demonstrating that autoregulation can compensate for the loss of one allele by increasing transcription of the remaining gene copy. Conversely, transgenic overexpression of Brn3a in the trigeminal ganglion suppresses the expression of the endogenous gene. These findings demonstrate that the Brn3a locus functions as a self-regulating unit to maintain a constant expression level of this key regulator of neural development.

The bulge region of the hair follicle serves as a repository for epithelial stem cells that can regenerate the follicle in each hair growth cycle and contribute to epidermis regeneration upon injury. Here we describe a population of multipotential stem cells in the hair follicle bulge region; these cells can be identified by fluorescence in transgenic nestin-GFP mice. The morphological features of these cells suggest that they maintain close associations with each other and with the surrounding niche. Upon explantation, these cells can give rise to neurosphere-like structures in vitro. When these cells are permitted to differentiate, they produce several cell types, including cells with neuronal, astrocytic, oligodendrocytic, smooth muscle, adipocytic, and other phenotypes. Furthermore, upon implantation into the developing nervous system of chick, these cells generate neuronal cells in vivo. We used transcriptional profiling to assess the relationship between these cells and embryonic and postnatal neural stem cells and to compare them with other stem cell populations of the bulge. Our results show that nestin-expressing cells in the bulge region of the hair follicle have stem cell-like properties, are multipotent, and can effectively generate cells of neural lineage in vitro and in vivo.

The POU-IV or Brn-3 class of transcription factors exhibit conserved structure, DNA-binding properties, and expression in specific subclasses of neurons across widely diverged species. In the mouse CNS, Brn-3.0 expression characterizes specific neurons from neurogenesis through the life of the cell. This irreversible activation of expression suggests positive autoregulation. To search for cis-acting elements that could mediate autoregulation we used a novel method, complex stability screening, which we applied to rapidly identify functional Brn-3.0 recognition sites within a large genomic region encompassing the mouse brn-3.0 locus. This method is based on the observation that the kinetic stability of Brn-3.0 complexes with specific DNA sequences, as measured by their dissociation half-lives, is highly correlated with the ability of those sequences to mediate transcriptional activation by Brn-3.0. The principal Brn-3.0 autoregulatory region lies ϳ5 kb upstream from the Brn-3.0 transcription start site and contains multiple Brn-3.0-binding sites that strongly resemble the optimal binding site for this protein class. This region also mediates transactivation by the closely related protein Brn-3.2, suggesting a regulatory cascade of POU proteins in specific neurons in which Brn-3.2 expression precedes Brn-3.0. Key words: Brn-3; POU-domain; autoregulation; transcription factor; homeodomain; dissociation kinetics; retina; habenula; inferior olive; tectumThe development of the vertebrate brain requires the differentiation of a large number of specific types of neurons from a relatively undifferentiated cell layer, the neural plate. Many developing neurons and their precursors express characteristic DNA-binding transcription factors. The expression domains of these factors overlap in unique combinations in specific regions of the neural tube and in groups of differentiating neurons, suggesting that development of each neuronal type is guided by a transcriptional code. However, little is known about how these factors act at the molecular level to determine and maintain specific neuronal phenotypes.Many of the transcription factors that are expressed in specific groups of neurons belong to the POU, Pax, and LIM families of variant homeodomain proteins. Some of these factors appear early in development in regions of the dividing neuroepithelium, whereas others are restricted to terminally differentiating neurons. Factors of the POU-IV or Brn-3 class are expressed as specific sets of C NS and peripheral sensory neurons exit the cell cycle (Fedtsova and T urner, 1995;Artinger et al., 1998), and they have been shown to be necessary for the normal development of the retina, auditory system, cranial sensory ganglia, and certain CNS nuclei (Ryan and Rosenfeld, 1997).The expression patterns of most of the neuron-specific transcription factors have been characterized only in the developing brain, and in many cases little information is available about their expression in the adult nervous system. Here we demonstrate that expressi...