Transcriptional Analysis of Melatonin-regulated Genes in the Sheep Pars Tuberalis using Next Generation Transcriptome Sequencing
Seasonally breeding mammals use photoperiod, encoded by rhythmical production of the nocturnal pineal hormone melatonin (MEL) as a critical cue to drive hormone rhythms and synchronise reproduction to the most optimal time of year.
Melatonin acts directly on specialised thyrotroph cells in the pars tuberalis (PT) of the pituitary, regulating expression of thyrotropin, such as TSH and TAC1, leading to seasonal neuroendocrine changes, which then relays messages back to the hypothalamus to control reproductive circuits. This process finally leads to seasonal neuroendocrine changes.
Next Generation Sequencing (NGS) as a novel high-throughput sequencing technology has been developed. One significant application of NGS is transcriptome sequencing, also known as RNA-seq. This allows researchers to measure transcription at an unprecedented throughput.
In the PT, melatonin induces Cry1 expression and recently we identified a sub-set of genes also induced by melatonin, suggesting that melatonin could regulate many pathways. With the aim of identifying the molecular control by melatonin in the PT, we undertook detailed gene expression analysis in this target organ using RNA-seq. RNAs were analysed from sheep treated with or without melatonin pellets. In this project, we aim to answer three questions. 1). what are the target genes regulated by melatonin? 2). what kind of genes are regulated? and 3). how do we think melatonin regulates genes, which includes: which targets are controlled by the melatonin regulated genes and how melatonin controls this.
Figure 1 Volcano Plot for genes which are directly or indirectly regulated by melatonin. Genes to the left are down-regulated and genes to the right up-regulated.
Using bioinformatic and statistical methods, we identified a total of 138 genes as significantly up-regulated by melatonin and 208 genes as significantly down-regulated within 1h of melatonin treatment. Several significantly expressed genes identified in this analysis are indicated in the Volcano plot (Figure 1).
Several genes (GTF2A1, NEUROD1, CRY1 and NAMPT) have previously been reported to be photoperiod and/or MEL regulated in the PT. More, novel genes have been detected as melatonin regulated genes in our experiment. We assigned the RNA-Seq reads mapping to the significantly expressed genes on the genome to see the exon and alternative splicing information.
Figure 2 shows an example of CRY1’s wiggle file. CRY1 is one gene identified as significantly up-regulated by melatonin with a false discovery rate of 5.65e-12 and 9.26-fold induction. These tracks (marked in red) plot all RNA-Seq read counts onto the CRY1 gene on the Bos Taurus reference genome. RNA-Seq read counts (marked in red) are displayed in windows of a set number of base pairs in width. From this figure, a number of peaks are observed in the melatonin treated situation which are barely visible in the un-treated situation.
The observed peaks reveal the locations of exons to be highly consistent with those predicted by Ensembl. The only exceptions are the boundaries for the 5’ and 3’UTRs. The 5’ and 3’ regions shown in this figure have been extended using the RNA-Seq data. RNA-seq therefore allows analysis at all levels: exon, transcript or gene.
Figure 2 Genome wiggle view of CRY1 showing RNA-seq tags mapped to individual exons regulated by MEL-treatment