Pineal Regulation: Neural, transsynaptic and intracellular control mechanisms

FRA-2/FOSL 2是一种碱性亮氨酸拉链结构域转录因子，广泛表达于哺乳动物组织中。该因子的功能库尚不清楚，部分原因是缺乏靶向基因组序列的知识。在这里，我们确定了新的，功能性FRA-2的目标基因组通过表达谱分析敲除转基因大鼠。在该模型中，松果体FRA-2的夜间节律被遗传编码的显性负突变蛋白抑制。经验证的FRA-2调节和非调节基因集的生物信息学分析表明，FRA-2调节子是有限的基因组靶选择规则，一般来说，超越核心顺式序列的身份。然而，一个变异AP-1相关（AP-1 R）序列是共同的一个子集的调控基因。候选AP-1 R序列的功能活性和蛋白质结合伴侣被确定为一种新的FRA-2抑制基因Rgs 4。FRA-2蛋白优先与近端Rgs 4 AP-1 R序列相关，如离体ChIP和体外EMSA分析所示;此外，转录抑制被AP-1 R序列的突变阻断，而上游共有AP-1家族序列的突变不影响Rgs 4表达。Rgs 4 AP-1 R序列蛋白复合物的夜间变化与辅激活因子CBP的FRA-2依赖性消除相关;这为Rgs 4基因抑制提供了机制基础。这些研究还提供了FRA-2选择性基因组靶向的功能见解，突出了预测和实际靶点之间的不一致性。未来的研究应该解决FRA-2-Rgs 4在其他系统中的相互作用，包括大脑，其中FRA-2功能知之甚少。(From Davies等人，2011年） AANAT转录的cAMP控制： 芳基烷基胺N-乙酰转移酶（AANAT）是控制褪黑激素生物合成日节律的关键调节酶。在鸡视网膜感光细胞中，Aanat转录和AANAT活性部分受cAMP依赖性机制调节。本研究的目的是确定负责cAMP依赖性诱导的鸡Aanat启动子内的调控元件。光感受器富集的视网膜细胞培养物用荧光素酶报告基因构建体转染，所述荧光素酶报告基因构建体含有高达4kb的5 '-侧翼区和Aanat的第一外显子。毛喉素处理刺激了由4kb启动子构建体和除最小的Aanat（-217至+120）luc之外的所有5 ′-缺失构建体驱动的荧光素酶活性。通过Aanat（-484至+120）luc构建体产生最大基础和毛喉素刺激的表达水平。这种结构缺乏一个典型的环状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：一个八次TTATT重复序列（TTATT（8））和一个CRE样序列。电泳迁移率变动测定，荧光素酶报告基因测定，染色质免疫沉淀和siRNA实验提供的证据表明，这些元素结合c-Fos，JunD和CREB，以增强基础和毛喉素刺激的Aanat转录。我们认为，在484 bp的近端启动子中的CRE样序列和TTATT（8）元件相互作用，介导了cAMP依赖的Aanat转录调控。(From Haque等人，2011）3） 膜电位控制：“用穿孔膜片钳记录法研究去甲肾上腺素对大鼠松果体细胞膜电位（V（m））和自发电活动的控制。去甲肾上腺素不改变尖峰频率。然而，发现它以浓度依赖性方式（0.1-10 m）通过（1B）-肾上腺素受体起作用，产生V（m）的双相变化。初始反应是由于瞬时（5秒）外向K（+）电流（50 pA）引起的超极化（从静息电位-46 mV到13 mV）。该电流似乎是由细胞内储存的Ca（2+）释放触发的，这是基于在Ca（2+）缺乏培养基中浸泡的细胞中也观察到的观察结果。此外，药理学研究表明，该电流依赖于磷脂酶C（PLC）激活，并部分由甲碘荷包牡丹碱和apamin敏感性Ca（2+）控制的K（+）通道介导。初始瞬时超极化之后，由于内向电流（10 pA）导致持续去极化（4 mV）。这种反应依赖于PLC依赖的Na（+）/Ca（2+）内流激活，但不涉及硝苯地平敏感的电压门控Ca（2+）通道。总之，这些结果首次表明，（1B）-肾上腺素受体的激活启动了松果体细胞V（m）的PLC依赖性双相变化，其特征是由Ca（2+）激活的K（+）通道的混合物介导的初始瞬时超极化，随后由Ca（2+）传导的非选择性阳离子通道介导的持续去极化。“（来自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)