Pineal Regulation: Neural, transsynaptic and intracellular control mechanisms

松果体调节：神经、突触和细胞内控制机制

• 批准号: 8736908
• 负责人: David Klein
• 金额: $ 23.04万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736908
• 关键词: 5' Flanking Region; Address; Affect; Anabolism; Apamin; Area; Arylalkylamine N-Acetyltransferase; Bathing; Binding; Bioinformatics; Biological Assay; Brain; CREB1 gene; Cardiovascular Physiology; Cations; Cell Culture Techniques; Cells; Chickens; Circadian Rhythms; Consensus; Controlled Study; Cyclic AMP; Cyclic AMP Response Element; Disease; Dominant-Negative Mutation; Dopamine; EMSA; Electrophoretic Mobility Shift Assay; Elements; Endocrine Physiology; Enzymes; Exons; Experimental Models; FOS gene; FOSL2 gene; Family; Forskolin; Fos-Related Antigens; Frequencies; Future; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Gland; Goals; Human; Human Biology; In Vitro; Indium; Intention; Knowledge; Leucine Zippers; Link; Luciferases; Mediating; Melatonin; Membrane; Membrane Potentials; Metabolism; Modeling; Molecular; Molecular Profiling; Mutation; Neurosecretory Systems; Nifedipine; Norepinephrine; Phospholipase C; Photoreceptors; Physiology; Pineal gland; Pinealocyte; Potassium Channel; Primates; Protein Binding; Proteins; Psyche structure; Rattus; Regulation; Regulatory Element; Regulon; Renal function; Reporter; Research; Retinal; Retinal Photoreceptors; Rodent; Role; Signal Transduction; Small Interfering RNA; System; Time; Tissues; Transcend; Transcription Factor AP-1; Transcriptional Regulation; Transgenic Organisms; Variant; Vertebrates; Work; base; bicuculline methiodide; cell type; chromatin immunoprecipitation; gene repression; improved; insight; nervous system disorder; neural stimulation; neuroregulation; novel; patch clamp; promoter; protein complex; receptor; relating to nervous system; research study; response; transcription factor; voltage

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)

FRA-2：FRA-2/FOSL2的作用是基本的 - 亮氨酸拉链基序转录因子，在哺乳动物组织中广泛表达。该因素的功能曲目尚不清楚，部分原因是缺乏针对性的基因组序列的知识。在这里，我们通过敲低转基因大鼠中的表达谱分析确定了整个基因组的新型功能性FRA-2靶标。在此模型中，松果体腺FRA-2的夜间节奏被遗传编码的主要阴性突变蛋白抑制。对经过验证的FRA-2调节和非调节基因的生物信息学分析表明，FRA-2调节受到基因组目标选择规则的限制，通常超越核心顺式顺式序列身份。然而，一个变体AP-1相关（AP-1R）序列对于调节基因的子集很常见。确定了候选AP-1R序列的功能活性和蛋白结合伙伴，用于新型FRA-2抑制基因RGS4。 FRA-2蛋白优先与近端RGS4 AP-1R序列相关，如离体芯片和体外EMSA分析所证明的。此外，转录抑制是通过AP-1R序列的突变阻断的，而上游共识AP-1家族序列的突变不会影响RGS4表达。 RGS4 AP-1R序列上蛋白质复合物的夜间变化与FRA-2依赖性解散CBP有关。这为RGS4基因抑制提供了机械基础。这些研究还提供了对FRA-2选择性基因组靶向的功能洞察力，突出了预测和实际靶标之间的不一致。未来的研究应解决其他系统（包括大脑）的FRA-2-RGS4相互作用，在该系统中，FRA-2功能的理解很少。 （摘自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的每日节奏的关键调节酶。在鸡视网膜感光细胞中，AANAT转录和AANAT活性部分受CAMP依赖机制调节。这项研究的目的是确定负责营地依赖性诱导的鸡肉AANAT启动子中的调节元素。富含光感受器的视网膜细胞培养物用荧光素酶报告基因构建体转染，该荧光素酶报告基因构建体，该构建体构建构建体，该构建体最多可容纳5'-Flanking区域和AANAT的第一个外显子。 Forskolin处理刺激了由4 KB启动子构建体驱动的荧光素酶活性，除最小的Aanat（-217至+120）Luc以外，所有5'Deption构建体刺激了荧光素酶活性。 AANAT（-484至+120）LUC构建体产生了最大的基底和福斯科蛋白刺激的表达水平。该构建体缺乏典型的循环AMP响应元件（CRE），但在其序列中包含另外两个潜在的重要元素：八次TTATT重复（TTATT（8））和CRE样序列。电泳迁移率转移测定，荧光素酶报告基因测定，染色质免疫沉淀和siRNA实验提供了证据，表明这些元素结合了C-FOS，JUND和CREB，以增强基础和福斯科林刺激的AANAT转录。我们建议484 bp近端启动子中的cre样序列和TTATT（8）元素相互作用，以介导依赖cAMP的转录调控AANAT。 （摘自Haque等人2011年）3） 控制膜电位的控制：“使用穿孔的斑块夹记录来研究膜电位（V（m））和自发性电活动的控制大鼠性肾上腺素。去甲肾上腺素。去甲肾上腺素并未改变尖峰频率。但是，它通过（1B） - 腺腺体浓度（1B）依赖性（1B）的浓度（0.1）依赖性（0.1）依赖性（0.1）依赖性（0.1）。 V（M）是由于临时（5秒）向外K（+）电流（50 pa）的超极化（从静止的势为-46 mV）。磷脂酶C（PLC）激活，部分由双核甲基二肽和呼吸暂停敏感的Ca（2+） - 受控的K（+）通道介导。该响应取决于Na（+）/Ca（2+）涌入的PLC依赖性激活，但不涉及硝苯地平敏感的电压门控CA（2+）通道。总共，这些结果首次表明（1b）肾上腺受体的激活引发了依赖PLC的双型双相变化，其特征是由CA（2+） - 活化的K（+）的混合介导的初始短暂性超极化介导的初始短暂性超极化，随后是通过ca（2+）介导的（2+）的（2+）（2+）（2+）。等2011年）