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 Ref 1. Circadian clock mechanism: Circadian clocks in vertebrates are thought to be composed of transcriptional-translational feedback loops involving a highly conversed set of 'clock genes'namely, period (Per1-3) and cryptochrome (Cry1-2), which encode negative transcriptional regulators;and Bmal1, Clock, and Npas2, which encode positive regulators. Aanat, which encodes arylalkylamine N-acetyltransferase (AANAT), the key regulatory enzyme that drives the circadian rhythm of melatonin synthesis, contains a circadian E-box element (CACGTG) in its proximal promoter that is potentially capable of binding CLOCK : BMAL1 and NPAS2 : BMAL1 heterodimers. The present study was conducted to investigate whether CLOCK and/or NPAS2 regulates Aanat expression in photoreceptor cells. Npas2 and Clock are both expressed in photoreceptor cells in vivo and in vitro. To assess the roles of CLOCK and NPAS2 in Aanat expression, gene-specific micro RNA vectors were used to knock down expression of these clock genes in photoreceptor-enriched cell cultures. The knockdown of CLOCK protein significantly reduced the circadian expression of Npas2, Per2, and Aanat transcripts but had no effect on the circadian rhythm of Bmal1 transcript level. The knockdown of NPAS2 significantly damped the circadian rhythm of Aanat mRNAs but had no effect on circadian expression of any of clock genes examined, except Npas2 itself. Chromatin immunoprecipitation studies indicated that both CLOCK and NPAS2 bound to the Aanat promoter in situ. Thus, CLOCK and NPAS2 have overlapping roles in the clock output pathway that regulates the rhythmic expression of Aanat in photoreceptors. However, CLOCK plays the predominant role in the chicken photoreceptor circadian clockwork mechanism, including the regulation of NPAS2 expression. (From 2) 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 3) Dopamine signal transduction: """"""""Dopamine plays diverse and important roles in vertebrate biology, impacting behavior and physiology through actions mediated by specific G-protein-coupled receptors, one of which is the dopamine receptor D4 (Drd4). Here we present studies on the >100-fold daily rhythm in rat pineal Drd4 expression. Our studies indicate that Drd4 is the dominant dopamine receptor gene expressed in the pineal gland. The gene is expressed in pinealocytes at levels which are approximately 100-fold greater than in other tissues, except the retina, in which transcript levels are similar. Pineal Drd4 expression is circadian in nature and under photoneural control. Whereas most rhythmically expressed genes in the pineal are controlled by adrenergic/cAMP signaling, Drd4 expression also requires thyroid hormone. This advance raises the questions of whether Drd4 expression is regulated by this mechanism in other systems and whether thyroid hormone controls expression of other genes in the pineal gland"""""""".(From 4) Control of membrane potential: """"""""Perforated patch clamp recording was used to study the control of membrane potential (V(m)) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through (1B)-adrenoreceptors in a concentration-dependent manner (0.1-10 m) to produce a biphasic change in V(m). The initial response was a hyperpolarization (13 mV from a resting potential of -46 mV) due to a transient (5 sec) outward K(+) current (50 pA). This current appears to be triggered by Ca(2+) released from intracellular stores, based on the observation that it was also seen in cells bathed in Ca(2+)-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive Ca(2+)-controlled K(+) channels. The initial transient hyperpolarization was followed by a sustained depolarization (4 mV) due to an inward current (10 pA). This response was dependent on PLC-dependent activation of Na(+)/Ca(2+) influx but did not involve nifedipine-sensitive voltage-gated Ca(2+) channels. Together, these results indicate for the first time that activation of (1B)-adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte V(m) characterized by an initial transient hyperpolarization mediated by a mixture of Ca(2+)-activated K(+) channels followed by a sustained depolarization mediated by a Ca(2+)-conducting nonselective cation channel."""""""" (From 5)

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Zip Code
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

Showing the most recent 10 out of 22 publications