Pineal Regulation: Developmental and Circadian Changes in the Transcriptome

全球基因表达分析：松果体基因表达特征的研究正在进行中。第一阶段涉及对大鼠松果体的分析：利用微阵列基因表达研究啮齿动物松果体转录组。比较中午和午夜的表达谱发现，全球1000个基因的表达变化了2倍，其中2/3在夜间增加。其中，400个基因的表达增加了4倍；器官培养的研究表明，在几乎所有的病例中，高度上调的基因的表达都是通过去甲肾上腺素或环核苷酸类似物诱导的。这些发现与NE-环核苷酸信号是导致夜间基因表达增加的主要机制相一致。然而，也很清楚还有其他机制参与其中，因为少数高节律基因不被去甲肾上腺素诱导或被弱诱导。将松果体中的基因表达水平与其他组织中的中位数进行比较，发现松果体中有一组&gt；300基因的表达水平是松果体的8倍。最高表达基因的一个重要子集编码与褪黑素合成和这一过程的控制有关的蛋白质，包括通过肾上腺素能受体和第二信使(包括环核苷酸、钙离子和磷脂)进行信号传递。高表达基因簇与甲状腺激素、维甲酸、谷氨酸等细胞生物学有关，与金属离子稳态、膜转运和免疫反应有关。其他高度和/或有节律性表达的基因也编码转录因子、离子通道、转运体、受体、调节分子和分泌产物，这些都是以前在松果体文献中没有出现过的。松果体基因表达谱与其他几个组织的比较进一步证明，松果体与视网膜最相似的是扩大了仅在这两个组织中高度表达的基因的数量。这项研究表明，松果体生物学的控制比之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之前认为的要多，并提供了分子证据来怀疑松果体可能在高度保守的褪黑素产生作用之外发挥作用。继对啮齿动物松果体的研究之后，还对猴子和人类的松果体进行了类似的研究，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序列技术进行扩展，除了注释基因外，还将重点放在miRNA和长的非编码RNA上。 长非编码RNA(摘自Coon等人，PNAS，2012)：长非编码RNA(LncRNAs)发挥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，我们提出了lncRNAs参与脊椎动物昼夜节律生物学的证据。112个lncRNAs(0.3~50kb)在夜间和白天的差异表达存在于大鼠的松果体中，松果体是夜间荷尔蒙褪黑激素的来源。这些变化中约有一半反映了夜间的增加。对8个具有2-to-gt；100倍每日节律的lncRNA的研究表明，在大多数情况下，这种变化是由视交叉上核的中央昼夜节律振荡器的神经刺激(加倍时间=0.5-1.3h)所致。夜间的光照会迅速逆转其中一些lncRNA的水平(减半时间=9-32分钟)。器官培养研究表明，这些lncRNAs的表达受去甲肾上腺素通过cAMP作用的调节。这些发现指出了lncRNAs在昼夜节律系统中的动态作用。 MicroRNAs：microRNAs(MiRNAs)在生物调节中发挥着广泛的作用。在这项研究中，首次对大鼠松果体miRNAs进行了描述，并通过关注松果体的主要功能褪黑素的合成来评估它们的重要性。下一代测序及相关方法表明，miRNA群体由一小群miRNAs主导：75%由10个miRNAs组成；miR-182占28%。除了miR-182，miR-183和miR-96在松果体中也高度丰富，这也是在视网膜中发现的一种独特的模式。这项工作还发现了以前无法识别的miRNAs和其他小的非编码RNA。松果体miRNA的丰度没有明显的黑夜/白昼差异，只有少数例外(例如，MiR-96和miR-182丰度的昼夜差异2倍)；这与松果体转录组的24小时动态模式形成鲜明对比。在发育过程中，大多数富含松果体的miRNAs的丰度增加；然而，至少有一种miR-483的丰度显著减少。MIR-483可能是褪黑素合成的调节因子，其基础如下：它抑制培养的松果体细胞褪黑素的合成；它通过芳基烷胺N-乙酰基转移酶(AANAT)3位基因为预测的结合位点而起作用；AANAT是褪黑素合成的倒数第二个酶；它表现出与AANAT转录本相反的发育特征。这些观察结果支持这样的假设，即miR-483在发育过程中抑制AANAT mRNA水平，而miR-483丰度的发育减少促进褪黑素的合成。

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.