Regulation of growth hormone receptor gene expression during development

To improve our understanding of the potential function of the human growth hormone receptor (hGHR) during fetal development, the ontogeny of hGHR mRNA expression in human tissues was examined. In addition, portions of the hGHR gene regulatory regions were cloned and characterized.Transcription of the hGHR gene was observed in multiple tissues from as early as the first trimester of human fetal life. Two isoforms of the mRNA coding region were identified, exon 3-retained or -deleted, and it was shown that expression of these two transcripts is individual-, but not tissue-, specific. In tissues from 117 individuals (4 weeks of fetal age to 64 years postnatal), predominant expression of the exon 3-deleted isoform occurred prior to 20 weeks of fetal life, suggesting that exon 3-deleted transcripts may be developmentally regulated. A six-fold increase in hepatic hGHR mRNA was observed postnatally, while the levels in kidney and intestine showed no significant developmental change and those in lung decreased six-fold postnatally.This suggested that multiple gene promoters might be directing tissue-specific developmental changes in hGHR mRNA levels. Since heterogeneity in the 5 ' untranslated regions (5'UTR) of mRNAs can result from differential gene promoter usage, the expression patterns of three of the eight known 5'UTR variants (VI to V8, numbered according to their relative abundance in liver) were then investigated. Expression of V1, V3 and V4 was compared in fetal versus postnatal tissues, including hepatoblastomas and hepatocellular carcinomas. While V3 was detected in all tissues examined, the V1 and V4 variants were only present in normal postnatal liver specimens; they were not expressed in hepatic tumours. It was hypothesized that VI and V4 are derived by alternative splicing of a common gene transcript, while V3 synthesis is regulated by a distant and more widely transcribed gene promoter.To identify DNA sequences regulating synthesis of the V1 and V4 5 'UTR variants, portions of the 5' flanking region of the hGHR gene were cloned. Four of the 5'UTR variant sequences were precisely placed in series (5' V7-V1-V4-V8 3') on a 3.8 kb XbaI-BsaAI genomic fragment. A transcriptional start site was identified upstream of the V1 sequence and found to generate, in postnatal liver, a long 5'UTR exon containing V1, V4 and V8 sequences. Evidence for a second liver-specific transcriptional start site, located upstream of V7, was also obtained. Alternative splicing of these gene products can result in several mRNA species, including the previously isolated V1, V4, V7 and V8 mRNA isoforms. Preliminary characterization of a 4.3 kb HindIII-XbaI genomic clone has revealed that the V3 as well as V2 sequences are located in series (5' V2-V3 3' ) in a distant genomic region, and computer analysis has suggested that there are transcriptional start sites upstream of both the V2 and V3 sequences. To construct a complete and continuous physical map of the hGHR gene, a Bacterial Artificial Chromosome (BAC) recombinant clone, encompassing more than 100 kb human genomic DNA and containing both V1- and V3-variant clusters, was isolated. Future characterization of the BAC clone will produce a complete map of the hGHR gene regulatory regions.In summary, developmental changes in hGHR gene expression most likely reflect the maturation of an endocrine system but may also confer fetal-specific hGHR biological activity