Pineal Regulation: Neural, transsynaptic and intracellular control mechanisms

FRA-2的作用：FRA-2/FOSL2是一个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。该因子的功能谱系尚不清楚，部分原因是缺乏对靶向基因组序列的了解。在这里，我们通过基因敲除转基因大鼠的表达谱分析，确定了整个基因组中新的、有功能的FRA-2靶点。在这个模型中，松果体FRA-2的夜间节律被一种遗传编码的显性负性突变蛋白抑制。对FRA-2调控和非调控基因的生物信息学分析表明，FRA-2调节子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同一性。然而，一个变异的AP-1相关(AP-1R)序列是调控基因的一个子集所共有的。对一个新的FRA-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进行了测定。体外芯片和体外EMSA分析表明，FRA-2蛋白优先与RGS4 AP-1R近端序列相关；此外，AP-1R序列的突变阻止了转录抑制，而上游共识AP-1家族序列的突变不影响RGS4的表达。RGS4 AP-1R序列蛋白质复合体的夜间变化与FRA-2依赖的共激活因子CBP的消除有关，这为RGS4基因抑制提供了机制基础。这些研究还为FRA-2选择性基因组靶向提供了功能洞察力，突出了预测靶点和实际靶点之间的不一致。未来的研究应该解决FRA-2和RGS4在其他系统中的相互作用，包括大脑，因为FRA-2的功能在大脑中知之甚少。(摘自Davies等人2011年) AANAT转录的cAMP调控：芳烷基胺N-乙酰基转移酶(AANAT)是控制褪黑素生物合成的关键调节酶。在鸡视网膜光感受器细胞中，AANAT转录和AANAT活性部分受cAMP依赖机制的调节。本研究的目的是确定鸡AANAT启动子中负责cAMP依赖诱导的调控元件。将含有长达4kb的5‘-侧翼区和AANAT第一外显子的荧光素酶报告结构导入感光细胞培养物中。Forsklin处理后，荧光素酶活性由4&8195；kb启动子和除最小的AANAT(-217到+120)Luc以外的所有5‘-缺失结构驱动。AANAT(-484到+120)LUC构建产生最大的基础和Forsklin刺激的表达水平。该结构缺乏典型的环状AMP反应元件(CRE)，但在其序列中包含另外两个潜在的重要元件：一个8倍TTATT重复(TTATT(8))和一个类似Cre的序列。电泳迁移率改变分析、荧光素酶报告分析、染色质免疫沉淀和siRNA实验证明，这些元件与c-Fos、Jund和CREB结合，以增强基础和Forsklin刺激的AANAT转录。我们认为，484&8195；bp启动子近端的Cre-like序列和TTATT(8)元件相互作用，介导cAMP依赖的AANAT转录调控。(摘自Haque等人，2011年) 膜电位的控制：“穿孔膜片钳记录研究去甲肾上腺素对松果体细胞膜电位(V(M))和自发性电活动的控制作用。去甲肾上腺素不改变放电频率。然而，它被发现通过(1B)-肾上腺素受体以浓度依赖的方式(0.1-10m)作用，产生V(M)的双相变化。最初的反应是由瞬间(5s)外向钾电流(50pA)引起的超极化(从-46 mV的静息电位到13 mV)。这一电流似乎是由细胞内钙离子释放触发的，基于观察到它也存在于沐浴在缺钙介质中的细胞中。此外，药理学研究表明，这一电流依赖于磷脂酶C(PLC)的激活，并部分由荷包牡丹碱碘和阿帕明敏感的Ca(2+)控制的K(+)通道介导。在最初的瞬时超极化之后，由于内向电流(10pA)，持续去极化(4 MV)。这种反应依赖于依赖PLC激活的Na(+)/Ca(2+)内流，但不涉及硝苯地平电压门控钙通道。综上所述，这些结果首次表明，(1B)-肾上腺素受体的激活在松果体细胞V(M)上启动了一种依赖PLC的双时相变化，其特征是最初由钙(2+)激活的K(+)通道的混合物介导的瞬时超极化，然后由钙(2+)传导的非选择性阳离子通道介导的持续去极化。(摘自Zemkova等人，2011年)

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)