Action of Fra-2: FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood. (From Davies et al 2011) cAMP control of AANAT transcription: Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the 4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT(8) ) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT(8) elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat. (From Haque et al 2011)3)

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Project End
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Support Year
9
Fiscal Year
2015
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U.S. National Inst/Child Hlth/Human Dev
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Yamazaki, Fumiyoshi; Møller, Morten; Fu, Cong et al. (2015) The Lhx9 homeobox gene controls pineal gland development and prevents postnatal hydrocephalus. Brain Struct Funct 220:1497-509
Yamazaki, Fumiyoshi; Kim, Hyun Hee; Lau, Pierre et al. (2014) pY RNA1-s2: a highly retina-enriched small RNA that selectively binds to Matrin 3 (Matr3). PLoS One 9:e88217
Falcón, Jack; Coon, Steven L; Besseau, Laurence et al. (2014) Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya. Proc Natl Acad Sci U S A 111:314-9
Kucka, Marek; Bjelobaba, Ivana; Clokie, Samuel J H et al. (2013) Female-specific induction of rat pituitary dentin matrix protein-1 by GnRH. Mol Endocrinol 27:1840-55
Matsuo, Masahiro; Coon, Steven L; Klein, David C (2013) RGS2 is a feedback inhibitor of melatonin production in the pineal gland. FEBS Lett 587:1392-8
Rath, Martin F; Rohde, Kristian; Klein, David C et al. (2013) Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance. Neurochem Res 38:1100-12
Ochocinska, Margaret J; Muñoz, Estela M; Veleri, Shobi et al. (2012) NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies. J Neurochem 123:44-59
Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee et al. (2012) MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase. J Biol Chem 287:25312-24
Davies, Jeff S; Klein, David C; Carter, David A (2011) Selective genomic targeting by FRA-2/FOSL2 transcription factor: regulation of the Rgs4 gene is mediated by a variant activator protein 1 (AP-1) promoter sequence/CREB-binding protein (CBP) mechanism. J Biol Chem 286:15227-39
Zemkova, Hana; Stojilkovic, Stanko S; Klein, David C (2011) Norepinephrine causes a biphasic change in mammalian pinealocye membrane potential: role of alpha1B-adrenoreceptors, phospholipase C, and Ca2+. Endocrinology 152:3842-51

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