MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.

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Phosphorylation of the transcription factor CREB leads to the recruitment of the coactivator, CREB binding protein (CBP). Recent studies have suggested that CBP recruitment is not sufficient for CREB function, however. We have identified a conserved protein-protein interaction motif within the CBP-binding domains of CREB and another transcription factor, SREBP (sterol-responsive element binding protein). In contrast to CREB, SREBP interacts with CBP in the absence of phosphorylation. We have exploited the conservation of this interaction motif to test whether CBP recruitment to CREB is sufficient for transcriptional activation. Substitution of six nonconserved amino acids from SREBP into the activation domain of CREB confers high-affinity, phosphorylation-independent CBP binding. The mutated CREB molecule, CREB DIEDML , activates transcription in F9 teratocarcinoma and PC12 cells even in the absence of protein kinase A (PKA). Addition of exogenous CBP augments the level of transcription mediated by CREB DIEDML , and adenovirus 12S E1A blocks transcription, implicating CBP in the activation process. Thus, recruitment of CBP to CREB is sufficient for transcriptional activation. Addition of PKA stimulates transcription induced by CREB DIEDML further, suggesting that a phosphorylation event downstream from CBP recruitment augments CREB signaling.The signaling mechanism that activates genes through the cyclic AMP (cAMP)-regulated enhancer (CRE) (23) represents one of the most intensively studied transcriptional pathways. This pathway consists of protein kinase A (PKA), the transcription factor CREB, and the coactivator CREB binding protein (CBP) (3,7,18). CBP has been shown to participate in many additional transcriptional pathways as well (11), but the mechanism by which it activates gene expression remains uncertain. CBP and its homologue p300 interact with the basal transcription factors TFIID and TFIIB, as well as with the RNA polymerase II holoenzyme component, RNA helicase A (9, 24, 34), suggesting that one function of this coactivator is to stabilize the preinitiation complex. Other studies have argued that transcriptional activation through CBP/p300 occurs only in the context of chromatin, however (17). The involvement of chromatin in CBP function is consistent with the finding that CBP, and several associated proteins including P/CAF, steroid receptor coactivator 1, and p/CIP, have the ability to acetylate the amino-terminal tails of histone proteins (6,31,36,38). These and other posttranslational modifications of chromatin components induced by the cAMP signaling cascade may stimulate transcription through nucleosome remodeling.Of all transcription factor-CBP associations, only the interaction with phosphorylated CREB has been characterized in detail. Our lab has studied this association by using a fluorescence polarization binding assay and a genetic interaction assay in yeast (18,30). These studies indicated that CREB phosphorylated at Ser 133 binds to CBP with an affinity of approximately 350 nM and that...

CtBP (carboxyl-terminal binding protein) participates in regulating cellular development and differentiation by associating with a diverse array of transcriptional repressors. Most of these interactions occur through a consensus CtBP-binding motif, PXDLS, in the repressor proteins. We previously showed that the CtBP-binding motif in E1A is flanked by a Lys residue and suggested that acetylation of this residue by the p300/CBPassociated factor P/CAF disrupts the CtBP interaction. In this study, we show that the interaction between CtBP and the nuclear hormone receptor corepressor RIP140 is regulated similarly, in this case by p300/CBP itself. CtBP was shown to interact with RIP140 in vitro and in vivo through a sequence, PIDLSCK, in the amino-terminal third of the RIP140 protein. Acetylation of the Lys residue in this motif, demonstrated in vivo by using an acetylated RIP140-specific antibody, dramatically reduced CtBP binding. Mutation of the Lys residue to Gln resulted in a decrease in CtBP binding in vivo and a loss of transcriptional repression. We suggest that p300/CBP-mediated acetylation disrupts the RIP140-CtBP complex and derepresses nuclear hormone receptor-regulated genes. Disruption of repressor-CtBP interactions by acetylation may be a general mode of gene activation.