Abstract
The effects of introns, especially the first intron, on the regulation of gene expression remains unclear. Therefore, the objective of the present study was to investigate the transcriptional regulatory function of intron 1 on the chicken growth hormone (cGH) gene in the rat pituitary tumor cell line (GH4-C1). Transient transfection using first-intron-inserted cGH complete coding sequences (CDSs) and non-intron-inserted cGH CDS plasmids, quantitative RT-PCR (qRT-PCR) and western blot assays were used to detect the expression of cGH. The reporter gene assay was also used to investigate the effect of a series of fragments in the first intron of cGH on gene expression in GH4-C1. All of the results revealed that a 200-bp fragment located in the +485/+684 region of intron 1 was essential for repressing the expression of cGH. Further informatics analysis showed that there was a cluster of 13 transcriptional factor binding sites (TFBSs) in the +485/+684 region of the cGH intron 1. Disruption of a glucocorticoid response-like element (the 19-nucleotide sequence 5′-AGGCTTGACAGTGACCTCC-3′) containing a T-box motif (TGACCT) located within this DNA fragment increased the expression of the reporter gene in GH4-C1. In addition, an electrophoretic mobility shift assay (EMSA) revealed a glucocorticoid receptor (GR) protein of rat binding to the glucocorticoid response-like element. Together, these results indicate that there is a negative glucocorticoid response-like element (nGRE) located in the +591/+609 region within the first intron of cGH, which is essential for the down-regulation of cGH expression.
Highlights
Introns, the first intron, have large positive or negative effects on the regulation of gene expression in mammalian and fish cells [1,2]
Analyses of this DNA fragment in the GH4-C1 cell lines from pituitary tissues of rat demonstrated that the expression of chicken growth hormone (cGH) was down-regulated by intron 1 on both the transcriptional and translational levels, which might result from a slower splicing reaction and regulatory element binding sites in intron 1 [37,38]
Consistent with a reduction in cGH protein by intron 1, the subsequent expressional experiments revealed markedly reduced reporter gene translational expression in GH4-C1, but this was not significantly regulated in transcriptional activity. This was further confirmed by the expressional profile of the +485/+684-reporter gene construct. These data indicated that the process of intron-splicing led to the repression of cGH transcriptional activity and that the +485/+684 region located in the first intron had a negative effect on the expression of cGH in vitro
Summary
The first intron, have large positive or negative effects on the regulation of gene expression in mammalian and fish cells [1,2]. In physiologically relevant cell lines, reporter gene assays on 107 genes in humans revealed that most regulatory elements were in the proximal regulatory promoter regions of the genes, which were located in the 5 -flanking and the intron 1 sequences [3]. Some of these first introns contained conserved sequences, which were the binding motifs of necessary functional transcription factors. When these regions were deleted by homologous recombination in knock-out mice, the mRNA level of a gene was diminished [4]. The function of dGRE was found to enhance the rat growth hormone (rGH) gene activity in the 5 -flanking sequences of rGH, similar to the role of glucocorticoid receptor cis-elements in the flanking region that could induce chicken growth hormone (cGH) gene expression in chicken embryonic pituitary cells [14,17,25]
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