Negative Glucocorticoid Response-Like Element from the First Intron of the Chicken Growth Hormone Gene Represses Gene Expression in the Rat Pituitary Tumor Cell Line.

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 () gene in the rat pituitary tumor cell line (GH4-C1). Transient transfection using first-intron-inserted complete coding sequences (CDSs) and non-intron-inserted CDS plasmids, quantitative RT-PCR (qRT-PCR) and western blot assays were used to detect the expression of . The reporter gene assay was also used to investigate the effect of a series of fragments in the first intron of 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 . Further informatics analysis showed that there was a cluster of 13 transcriptional factor binding sites (TFBSs) in the +485/+684 region of the 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 , which is essential for the down-regulation of expression.