Pineal Regulation: Neural, transsynaptic and intracellular control mechanisms

Fra-2的作用：Fra-2 /FOSL2是一种基本的区域亮氨酸拉链基序转录因子，在哺乳动物组织中广泛表达。该因子的功能库尚不清楚，部分原因是缺乏对目标基因组序列的了解。在这里，我们通过基因敲低转基因大鼠的表达谱分析，在基因组中发现了新的功能性fr -2靶点。在这个模型中，松果体fr -2的夜间节律被一种遗传编码的显性负突变蛋白抑制。对已验证的FRA-2调控和非调控基因集的生物信息学分析显示，FRA-2调控受到基因组靶标选择规则的限制，这些规则通常超越核心顺式序列身份。然而，一种变异ap -1相关（AP-1R）序列在一部分受调节基因中是共同的。测定了一种新的fr -2抑制基因Rgs4的候选AP-1R序列的功能活性和蛋白结合伙伴。体外ChIP和体外EMSA分析证实，fr -2蛋白优先与近端Rgs4 AP-1R序列相关；此外，AP-1R序列的突变阻断了转录抑制，而上游共识的AP-1家族序列的突变不影响Rgs4的表达。Rgs4 AP-1R序列蛋白复合物的夜间变化与fr -2依赖性共激活物CBP的解除有关；这为Rgs4基因抑制提供了机制基础。这些研究也为fr -2的选择性基因组靶向提供了功能见解，突出了预测目标与实际目标之间的不一致。未来的研究应该解决在其他系统中，包括大脑中，对fr -2功能知之甚少的fr -2- rgs4的相互作用。（摘自Davies et al 2011）

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) 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 Zemkova et al 2011)