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)

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)