Role of Sp Family Transcription Factors in Growth Hormone Receptor Gene Expression

Shin, Jeong Won; Yu, Jae Hong; An, Ren Zhe

Abstract

PURPOSE: The growth hormone receptor(GHR) is essential for the actions of growth hormone on postnatal growth and metabolism. GHR transcripts are characterized by the presence of disparate 5'untranslated exons. In contrast to L1 transcript, factors regulating the expression of the GC rich L2 transcript have remained unidentified. The purpose of this study is in order to characterize the mechanisms regulating expression of the L2 transcript in the murine GHR gene
METHODS
Transient transfection experiments including deletional analysis and co-transfection assay were performed to find a region containing promoter activity in the L2 5'flanking sequence using BNCL2(mouse liver) cells, CV-1(African green monkey kidney) cells, HRP.1 trophoblasts and Drosophila Schneider(SL2) cells. Sequencing analysis was performed to find the region contained consensus binding sites for transcription factors. Standard gel shift(Electrophoretic mobility shift assay, EMSA) and supershift analysis using liver nuclear extracts was performed to establish proteins(transcription factors) bound this regulatory element.
RESULTS
The 5'flanking region of the L2 untranslated region(UTR) exhibited promoter activity in BNCL2(mouse liver), CV-1(monkey kidney) cells and HRP.1 trophoblasts. Deletional analyses indicated the presence of a Sp binding site important for transcription of the L2 UTR and localized the major regulatory region within 75 bp of the 5'transcription start site. Sequencing analyses revealed the region contained consensus binding sites for the Sp family of transcription factors. EMSA and supershift EMSA revealed that in mouse liver nuclear extracts that Spl and Sp3 bound to this cis-element. Functional studies in Drosophila SL2 cells and BNCL2(mouse liver) cells established the ability of Sp3 and Sp1 to stimulate transcriptional activity via this cis-element. Functional studies in Drosophila SL2 cells demonstrated a functional interaction between Sp3 and Sp1 at this DNA-binding site.
CONCLUSION
Sp family transcription factors play a role in regulation of L2 transcript gene expression in the 5'flanking region of the murine GHR gene.