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. Neural Control of the rat pineal transcriptome(From Plus One 2015, in press).The term neurotranscriptomics is used here to describe genome-wide analysis of neural control of transcriptomes. In this report, next-generation RNA sequencing was using to analyze the effects of neonatal (5-days-of-age) surgical stimulus deprivation on the adult rat pineal transcriptome. In intact animals, more than 3000 coding genes were found to exhibit differential expression (adjusted-p < 0.001) on a night/day basis in the pineal gland (70% of these increased at night, 376 genes changed more than 4-fold in either direction). Of these, more than two thousand genes were not previously known to be differentially expressed on a night/day basis. The night/day changes in expression were almost completely eliminated by neonatal removal (SCGX) or decentralization (DCN) of the superior cervical ganglia (SCG), which innervate the pineal gland. Other than the loss of rhythmic variation, surgical stimulus deprivation had little impact on the abundance of most genes; of particular interest, expression levels of the melatonin-synthesis-related genes Tph1, Gch1, and Asmt displayed little change (less than 35%) following DCN or SCGX. However, strong and consistent changes were observed in the expression of a small number of genes including the gene encoding Serpina1, a secreted protease inhibitor that might influence extracellular architecture. Many of the genes that exhibited night/day differential expression in intact animals also exhibited similar changes following in vitro treatment with norepinephrine, a superior cervical ganglia transmitter, or with an analog of cyclic AMP, a norepinephrine second messenger in this tissue. These findings are of significance in that they establish that the pineal-defining transcriptome is established prior to the neonatal period. Further, this work expands our knowledge of the biological process under neural control in this tissue and underlines the value of RNA sequencing in revealing how neurotransmission influences cell biology. 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.
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