María Estela Andrés

PIASγ Represses the Transcriptional Activation Induced by the Nuclear Receptor Nurr1

Nurr1 is a transcription factor essential for the development of ventral dopaminergic neurons. In search for regulatory mechanisms of Nurr1 function, we identified the SUMO (small ubiquitin-like modifier)-E3 ubiquitin-protein isopeptide ligase, PIASγ, as an interaction partner of Nurr1. Overexpressed PIASγ and Nurr1 co-localize in the nuclei of transfected cells, and their interaction is demonstrated through co-immunoprecipitation and glutathione S-transferase pulldown assays. Co-expression of PIASγ with Nurr1 results in a potent repression of Nurr1-dependent transcriptional activation of an artificial NGFI-B response element (NBRE) reporter as well as of a reporter driven by the native tyrosine hydroxylase promoter. We identified two consensus sumoylation sites in Nurr1. The substitution of lysine 91 by arginine in one SUMO site enhanced the transcriptional activity of Nurr1, whereas the substitution of lysine 577 by arginine in the second SUMO site decreased transcriptional activity of Nurr1. Interestingly, PIASγ-induced repression of Nurr1 activity does not require the two sumoylation sites, because each mutant is repressed as efficiently as the wild type Nurr1. In addition, the mutations do not alter Nurr1 nuclear localization. Finally, we provide evidence that Nurr1 and PIASγ co-exist in several nuclei of the rodent central nervous system by demonstrating the co-expression of Nurr1 protein and PIASγ mRNA in the same cells. In conclusion, our studies identified PIASγ as a transcriptional co-regulator of Nurr1 and suggest that this interaction may have a physiological role in regulating the expression of Nurr1 target genes.

Estrogen Receptors α and β Differentially Regulate the Transcriptional Activity of the Urocortin Gene

Urocortin (Ucn), a highly conserved metazoan gene, is related to stress and feeding, behaviors with significant gender differences. We investigated whether estrogens regulate the expression of the Ucn gene using transient transfection in PC12 cells with the human Ucn (hUcn) promoter coupled to luciferase and either α or β estrogen receptors (ERα or ERβ, respectively). The results demonstrate that estradiol (E2) increases the activity of the hUcn promoter via ERα, and decreases hUcn promoter activity through ERβ. Deletions of the hUcn promoter show that the increase in promoter activity mediated by E2-ERα depends on a promoter region containing a half-estrogen response element and an Sp1 site, and the decrease mediated by E2-ERβ depends on a proximal promoter region containing a cAMP response element. Ucn and ERs coexist in neurons of rat hypothalamic nuclei, giving anatomical support for a direct effect of estrogen receptors on the Ucn gene. By in situ hybridization, we observed that cycling female rats have a higher number of cells expressing Ucn mRNA than males in the paraventricular nucleus of the hypothalamus (PVN) and the septum. Both of these brain nuclei are related to stress behaviors and express moderate levels of Ucn. Furthermore, Ucn mRNA was significantly decreased in the PVN and increased in the septum 30 d after ovariectomy. Acute E2 administration to ovariectomized rats significantly increased Ucn mRNA expression in the PVN and septum. In conclusion, our in vitro and in vivo evidence suggests that estrogens exert a direct and differential transcriptional regulation of the Ucn gene.

CoREST Represses the Heat Shock Response Mediated by HSF1

The stress response in cells involves a rapid and transient transcriptional activation of stress genes. It has been shown that Hsp70 limits its own transcriptional activation functioning as a corepressor of heat shock factor 1 (HSF1) during the attenuation of the stress response. Here we show that the transcriptional corepressor CoREST interacts with Hsp70. Through this interaction, CoREST represses both HSF1-dependent and heat shock-dependent transcriptional activation of the hsp70 promoter. In cells expressing short hairpin RNAs directed against CoREST, Hsp70 cannot repress HSF1-dependent transcription. A reduction of CoREST levels also provoked a significant increase of Hsp70 protein levels and an increase of HSF1-dependent transactivation of hsp70 promoter. Via chromatin immunoprecipitation assays we show that CoREST is bound to the hsp70 gene promoter under basal conditions and that its binding increases during heat shock response. In conclusion, we demonstrated that CoREST is a key regulator of the heat shock stress response.

Nurr1 regulates RET expression in dopamine neurons of adult rat midbrain.

J. Neurochem. (2010) 114, 1158–1167.

Nur transcription factors in stress and addiction

The Nur transcription factors Nur77 (NGFI-B, NR4A1), Nurr1 (NR4A2), and Nor-1 (NR4A3) are a sub-family of orphan members of the nuclear receptor superfamily. These transcription factors are products of immediate early genes, whose expression is rapidly and transiently induced in the central nervous system by several types of stimuli. Nur factors are present throughout the hypothalamus-pituitary-adrenal (HPA) axis where are prominently induced in response to stress. Drugs of abuse and stress also induce the expression of Nur factors in nuclei of the motivation/reward circuit of the brain, indicating their participation in the process of drug addiction and in non-hypothalamic responses to stress. Repeated use of addictive drugs and chronic stress induce long-lasting dysregulation of the brain motivation/reward circuit due to reprogramming of gene expression and enduring alterations in neuronal function. Here, we review the data supporting that Nur transcription factors are key players in the molecular basis of the dysregulation of neuronal circuits involved in chronic stress and addiction.

Rapid Increase of Nurr1 Expression in the Substantia Nigra After 6-Hydroxydopamine Lesion in the Striatum of the Rat

Nurr1 is a transcription factor essential for the genesis of ventral dopaminergic neurons. In this study, we investigated the expression of Nurr1 protein and mRNA in the adult rat brain by using immunohistochemistry and in situ hybridization, respectively. Another aim of our study was to investigate Nurr1 expression in substantia nigra after dopamine depletion induced by the injection of 6‐hydroxydopamine in the striatum. We observed that Nurr1 mRNA and protein are expressed in several brain regions, including cortex, hippocampus, substantia nigra, and ventral tegmental area, in agreement with previous reports using in situ hybridization. Additionally, we found that Nurr1 is expressed in brain regions that have not been previously reported, such as striatum, septum, and superior colliculus. Highest levels of expression were found in cortex, medial septum, dentate gyrus, some hypothalamic nuclei, and substantia nigra. Interestingly, we observed that, in the superior colliculus, Nurr1 protein is localized in the cytoplasm of cells, whereas, in other regions, it was localized mainly in the nuclei, suggesting that Nurr1 subcellular localization is regulated and may have functional implications. Dopamine depletion induced by an injection of 6‐hydroxydopamine into the striatum produced an increase in the number of cells expressing Nurr1 mRNA and protein in both substantia nigra compacta and substantia nigra reticulata, ipsilateral and contralateral to the lesioned side, measured 24 hr after the 6‐hydroxydopamine injection. These results suggest that Nurr1 may be involved in many neuronal functions in the adult central nervous system and, in particular, might be related to the compensation processes that take place in dopaminergic cells in order to normalize extracellular dopamine levels in the striatum.

Differential Properties of Transcriptional Complexes Formed by the CoREST Family

ABSTRACT Mammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1), and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases 1 and 2 (HDAC1/2) and the finding that all three CoRESTs are expressed in the adult rat brain. CoRESTs interact equally strongly with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2, which is barely present in LSD1/KDM1A-CoREST2 complexes. A nonconserved leucine in the first SANT domain of CoREST2 severely weakens its association with HDAC1/2. Furthermore, CoREST2 mutants with increased HDAC1/2 interaction and those without HDAC1/2 interaction exhibit equivalent transcriptional repression capacities, indicating that CoREST2 represses in an HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental in the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine-tuning gene expression regulation.

CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface.

Significance Corticotropin releasing factor binding protein (CRF-BP) belongs to the CRF family that is fundamental in the stress response and in the interaction between stress and addiction. The mechanisms by which CRF-BP regulates the CRF system are not fully understood. Most G protein-coupled receptors (GPCRs) are located mainly intracellularly and depend on specific escort proteins for their trafficking to the cell surface. CRF2αR is also located intracellularly; however, no escort protein regulating its presence on the cell surface has been described. We show that CRF-BP interacts with CRF2αR, acting as an escort protein, increasing the presence of the receptor on the cell surface. Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF2R). CRF2R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF2R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF2R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF2αR in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF2R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF2αR in the endoplasmic reticulum. In contrast, CRF-BP and CRF2αR colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF2αR but not the CRF2βR isoform, increasing CRF2αR on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.

Activation of GABA-B receptors induced by systemic amphetamine abolishes dopamine release in the rat lateral septum

J. Neurochem. (2010) 114, 1678–1686.

PIASγ enhanced SUMO-2 modification of Nurr1 activation-function-1 domain limits Nurr1 transcriptional synergy.

Nurr1 (NR4A2) is a transcription factor that belongs to the orphan NR4A group of the nuclear receptor superfamily. Nurr1 plays key roles in the origin and maintenance of midbrain dopamine neurons, and peripheral inflammatory processes. PIASγ, a SUMO-E3 ligase, represses Nurr1 transcriptional activity. We report that Nurr1 is SUMOylated by SUMO-2 in the lysine 91 located in the transcriptional activation function 1 domain of Nurr1. Nurr1 SUMOylation by SUMO-2 is markedly facilitated by overexpressing wild type PIASγ, but not by a mutant form of PIASγ lacking its first LXXLL motif (PIASγmut1). This PIASγmut1 is also unable to interact with Nurr1 and to repress Nurr1 transcriptional activity. Interestingly, the mutant PIASγC342A that lacks SUMO ligase activity is still able to significantly repress Nurr1-dependent transcriptional activity, but not to enhance Nurr1 SUMOylation. A SUMOylation-deficient Nurr1 mutant displays higher transcriptional activity than the wild type Nurr1 only in promoters harboring more than one Nurr1 response element. Furthermore, lysine 91, the major target of Nurr1 SUMOylation is contained in a canonical synergy control motif, indicating that SUMO-2 posttranslational modification of Nurr1 regulates its transcriptional synergy in complex promoters. In conclusion, PIASγ can exert two types of negative regulations over Nurr1. On one hand, PIASγ limits Nurr1 transactivation in complex promoters by SUMOylating its lysine 91. On the other hand, PIASγ fully represses Nurr1 transactivation through a direct interaction, independently of its E3-ligase activity.