Pineal Regulation: Developmental and Circadian Changes in the Transcriptome

全球基因表达的分析：研究正在进行中，其特点是在松果体中的基因表达。 第一阶段涉及大鼠松果体的分析： 啮齿动物松果体转录组研究使用微阵列基因表达。 午间和午夜表达谱的比较显示，1000个基因的表达总体变化>2倍，其中2/3在夜间增加。 其中，400个表达增加>4倍;器官培养中的研究揭示，在几乎所有情况下，高度上调的基因的表达通过用NE或环核苷酸类似物处理来诱导。 这些发现与NE-环核苷酸信号传导是夜间基因表达增加的主要机制的结论一致。 然而，也很明显，其他机制参与，因为少数高度节律的基因不诱导或弱诱导NE处理。 将松果体中的基因表达水平与其他组织中的中值表达进行比较，表明一组> 300个基因在松果体中的表达高出>8倍。 最高表达基因的重要子集编码参与褪黑激素合成和该过程的控制的蛋白质，包括通过肾上腺素能受体和第二信使（包括环核苷酸、Ca++和磷脂）的信号传导。 高表达基因簇与甲状腺激素、维甲酸、谷氨酸生物学的细胞生物学相关;并且与金属离子稳态、膜运输和免疫应答相关。 其他高度和/或节律性表达的基因也编码转录因子，离子通道，转运蛋白，受体，调节分子和分泌产物，以前没有出现在松果体文献。 比较松果体基因表达谱的几个其他组织增加的证据表明，松果体是最相似的视网膜，通过扩大的基因的数量，高度表达专门在这两个组织。 这项研究表明，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认为的要高，也提供了分子证据来怀疑松果体可能在褪黑激素产生中发挥高度保守的作用。 在啮齿动物松果体上的工作之后，正在对猴子和人类的松果体进行类似的工作，以确定这三种组织中基因表达模式的相似性。 这项工作正在使用RNA Seq技术进行扩展，除了注释基因外，还专注于miRNA和长非编码RNA。 长链非编码RNA（来自Coon等人，PNAS，2012）：长链非编码RNA（lncRNA）发挥广泛的生物学作用，包括调控基因和染色体的表达。在这里，我们提出的证据表明，lncRNA参与脊椎动物的昼夜节律生物学。112种lncRNA（0.3至>50 kb）的夜间/白天差异表达发生在大鼠松果体中，松果体是褪黑激素（夜间激素）的来源。这些变化中约有一半反映了夜间的增加。对8种具有2倍至>100倍日节律的lncRNA的研究表明，在大多数情况下，这种变化是由视交叉上核中的中央昼夜节律振荡器的神经刺激引起的（倍增时间= 0.5-1.3 h）。夜间的光暴露迅速逆转（减半时间= 9-32分钟）这些lncRNA中的一些的水平。器官培养研究表明，这些lncRNA的表达是由去甲肾上腺素通过cAMP作用调节的。这些发现指出lncRNA在昼夜节律系统中的动态作用。 MicroRNA：MicroRNA（miRNAs）在生物调节中发挥着广泛的作用。在这项研究中，大鼠松果体的miRNAs的第一次和他们的重要性进行了评估，集中在松果体的主要功能，褪黑激素的合成。下一代测序和相关方法揭示了miRNA群体由一小群miRNA主导：75%由10种miRNA占; miR-182占28%。除了miR-182之外，miR-183和miR-96也在松果体中高度富集，这是在视网膜中也发现的独特模式。这项工作还确定了以前未被识别的miRNA和其他小的非编码RNA。松果体miRNAs在丰度上没有表现出明显的昼夜差异，只有少数例外（例如，2-miR-96和miR-182丰度的夜/日差异倍）;这与松果体转录组特征的动态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的丰度增加;然而，至少有一种miR-483显著减少。miR-483是褪黑激素合成的可能调节剂，基于以下：它抑制培养物中松果体细胞的褪黑激素合成;它通过预测的芳基烷基胺N-乙酰转移酶（Aanat）（褪黑激素合成中的倒数第二个酶）的3-prime UTR中的结合位点起作用;并且，它表现出与Aanat转录物相反的发育概况。这些观察结果支持了这样的假设：miR-483在发育过程中抑制Aanat mRNA水平，并且miR-483丰度的发育减少促进褪黑激素合成。

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.