Pineal Regulation: Developmental and Circadian Changes in the Transcriptome

松果体调节：转录组的发育和昼夜节律变化

• 批准号: 8553949
• 负责人: David Klein
• 金额: $ 47.09万
• 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/8553949
• 关键词: 5' Flanking Region; Accounting; Acids; Adrenergic Receptor; Area; Arylalkylamine N-Acetyltransferase; Binding Sites; Biological; Biology; Cellular biology; Chromosomes; Circadian Rhythms; Complex; Cyclic AMP; Cyclic Nucleotides; Data; Development; Enzymes; Equipment and supply inventories; Exhibits; Functional RNA; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Gland; Glutamates; Homeostasis; Hormones; Hour; Human; Immune response; Ion Channel; Ions; Knowledge; Light; Literature; Melatonin; Membrane Protein Traffic; Messenger RNA; Metabolic Pathway; Metals; Methods; MicroRNAs; Modeling; Molecular; Molecular Profiling; Monkeys; Norepinephrine; Organ Culture Techniques; Pattern; Phenotype; Phospholipids; Pineal gland; Pinealocyte; Play; Population; Process; Production; Proteins; RNA; RNA Splicing; Rattus; Regulation; Retina; Retinoids; Rodent; Role; Second Messenger Systems; Signal Transduction; Site; Source; Staging; System; Technology; Thyroid Hormones; Time; Tissues; Transcript; Untranslated RNA; Untranslated Regions; Work; base; follow-up; neural stimulation; next generation; nucleotide analog; receptor; second messenger; suprachiasmatic nucleus; transcription factor

Analysis of global gene expression: Studies are in progress which have characterized gene expression in the pineal gland. The first stage has involved analysis of the rat pineal gland: The rodent pineal transcriptome was investigated using microarray gene expression. Comparison of midday and midnight expression profiles revealed that a global >2-fold change in the expression of 1000 genes, 2/3 of which increase at night. Among these, 400 increase >4- fold in expression; studies in organ culture reveal that in nearly all cases, the expression of the highly upregulated genes is induced by treatment with NE or cyclic nucleotide analogs. These findings are consistent with the conclusion that NE-cyclic nucleotide signaling is the primary mechanism responsible for the nocturnal increase in gene expression. However, it is also clear that other mechanisms are involved, because a small number of highly rhythmic genes are not induced or are weakly induced by NE treatment. Comparison of the level of gene expression in the pineal gland to the median expression in other tissues indicates that a set of > 300 genes are expressed >8- fold higher in the pineal gland. A significant subset of the most highly expressed genes encode proteins involved in melatonin synthesis and the control of this process, including signalling via adrenergic receptors and second messengers including cyclic nucleotides, Ca++ and phospholipids. Clusters of highly expressed genes are associated with the cellular biology of thyroid hormone, retinoid acid, glutamate biology; and, with metal ion homeostasis, membrane trafficking, and the immune response. Other highly and/or rhythmically expressed genes also encode transcription factors, ion channels, transporters, receptors, regulatory molecules and secreted products that have not previously appeared in the pineal literature. Comparison of the pineal gene expression profile to that of several other tissues adds to the evidence that the pineal gland is most similar to the retina by expanding the number of genes that are highly expressed exclusively in these two tissues. This study indicates that control of pineal biology is significantly more complex than previously thought, that the number of highly expressed genes in the pineal gland and retina is higher than previously thought, and also provides molecular evidence to suspect that the gland might function outside of the highly conserved role it plays in melatonin production. The work on the rodent pineal gland is being followed up with similar work on the pineal gland of the monkey and human, so as to determine the similarity of the patterns of gene expression in these three tissues. This work is being extended using RNA Seq technology, with focus on miRNA and long noncoding RNAs in addition to annotated genes. Long noncoding RNAs (From Coon et al, PNAS, 2012): Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system. MicroRNAs: MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study rat pineal miRNAs were profiled for the first time and their importance evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Next-generation sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs: 75% is accounted for by 10 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small non-coding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (eg. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-hour pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following: it inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3-prime UTR of arylalkylamine N-acetyltransferase (Aanat), the penultimate enzyme in melatonin synthesis; and, it exhibits a developmental profile opposite to that of Aanat transcripts. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

全球基因表达的分析：正在进行的研究中，这些研究表征了松果体中基因表达。 第一阶段涉及对大鼠松果体的分析：使用微阵列基因表达研究了啮齿动物松果体转录组。 中午和午夜表达曲线的比较表明，1000个基因表达的全球> 2倍变化，其中2/3在夜间增加。 其中，表达增加了400倍> 4倍；器官培养中的研究表明，在几乎所有情况下，高度上调的基因的表达是通过NE或环状核苷酸类似物处理诱导的。 这些发现与结论是一致，即NE-循环核苷酸信号传导是导致基因表达夜间增加的主要机制。 但是，很明显，还涉及其他机制，因为少数高度节奏的基因未诱导或通过NE治疗弱诱导。 松果体中基因表达水平与其他组织中的中位表达的比较表明，在松果体中，一组> 300个基因表达> 8倍。 最高表达基因的显着子集编码涉及褪黑激素合成的蛋白质以及对该过程的控制，包括通过肾上腺素能受体和包括环状核苷酸，CA ++和磷脂的第二个使者信号传导。 高度表达基因的簇与甲状腺激素，视视视感素酸，谷氨酸生物学的细胞生物学有关。并具有金属离子稳态，膜运输和免疫反应。 其他高度和/或有节奏表达的基因还编码转录因子，离子通道，转运蛋白，受体，调节分子和分泌的产物，这些产品以前尚未出现在松果文献中。 松果基因表达谱与其他几个组织的比较增加了通过扩大在这两种组织中仅高度表达的基因的数量，这表明松果体与视网膜最相似。 这项研究表明，对松果体生物学的控制比以前认为的要复杂得多，松果体和视网膜中高表达基因的数量比以前想象的要高，并且还提供了分子证据，以怀疑该腺可能在褪黑激素产生中发挥的高度保守的作用。 在啮齿动物和人类的松果体上进行了类似的工作，在啮齿动物松果体上的工作进行了跟踪，以确定这三个组织中基因表达模式的相似性。 这项工作是使用RNA SEQ技术扩展的，除了注释的基因外，还针对miRNA和长期非编码RNA。 长的非编码RNA（来自Coon等，PNA，2012年）：长的非编码RNA（LNCRNA）扮演着广泛的生物学作用，包括调节基因和染色体的表达。在这里，我们提供了证据，表明lncRNA参与脊椎动物昼夜节律生物学。在大鼠松果体中发生了112 lncRNA（0.3至> 50 kb）的差异夜间/白天表达，这是褪黑激素的来源，这是夜晚的激素。这些变化的大约一半反映了夜间的增加。对八个具有2至100倍每日节律的LNCRNA的研究表明，在大多数情况下，这种变化是由于昼夜节律核核中心振荡器的神经刺激而导致的（倍增时间= 0.5-1.3 h）。夜间的光线暴露迅速逆转（将时间= 9-32分钟）的某些LNCRNA水平。器官培养研究表明，这些LNCRNA的表达受到通过cAMP作用的去甲肾上腺素调节。这些发现表明LNCRNA在昼夜节律系统中的动态作用。 microRNA：microRNA（miRNA）在生物调节中起着广泛的作用。在这项研究中，首次对大鼠松果体miRNA进行了介绍，并通​​过关注松果体的主要功能，褪黑激素合成来评估其重要性。下一代测序和相关方法表明，miRNA群体由一小部分miRNA主导：75％由10个miRNA占。 miR-182代表28％。除miR-182外，miR-183和miR-96在松果体中也高度富集，在视网膜中也发现了一种独特的模式。这项工作还确定了先前未识别的miRNA和其他小型非编码RNA。松果体miRNA在丰度中没有明显的夜晚差异（例如，Mir-96和miR-182的丰度差异2倍夜/天差异）；这与特征松果体转录组的动态24小时模式形成鲜明对比。在发育过程中，大多数富含松果体的miRNA的丰富度增加了。但是，至少一个miR-483有明显的减少。 miR-483可能是基于以下情况的褪黑激素合成的调节剂：它抑制培养基中松果体的褪黑激素合成；它通过预测的芳基烷基N-乙酰基转移酶（AANAT）的3杆UTR中的预测结合位点起作用，这是褪黑激素合成中的倒数第二个酶；而且，它表现出与AANAT转录本相反的发展概况。这些观察结果支持miR-483在发育过程中抑制AANAT mRNA水平的假设，而miR-483丰度的发育降低会促进褪黑激素的合成。