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，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）

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)