Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon

Cheng-Ming Lee,Yu-Zen Huang,Tai-Jay Chang,Chia-Hung Yen,Kuan-Hsien Chou,Tsai-Yu Tzeng,Yi-Ming Arthur Chen

doi:10.1042/bsr20130030

Abstract

Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.

Highlights

prostate cancer (PCa) is the third most common cancer for men throughout the planet, and the most common among men in North America, Europe and some parts of Africa [1]
The results showed that glycine N-methyltransferase (GNMT) is an androgen-inducing gene and a functional androgen response element (ARE) is located in the coding region of the exon 1
The androgen agonist-R1881 induces GNMT expression in PCa cells To investigate the relationship between androgen stimulation and GNMT expression, LNCaP cells that express androgen receptor (AR) were treated with R1881 for 24 h

Summary

Introduction

PCa (prostate cancer) is the third most common cancer for men throughout the planet, and the most common among men in North America, Europe and some parts of Africa [1]. Androgen plays a crucial role in the pathogenesis of PCa [3,4]. The activated AR (androgen receptor) is translocated into the nucleus where it binds to AREs (androgen response elements) present in different target genes [5]. GNMT (glycine N-methyltransferase) affects epigenetic modification by regulating the ratio of SAM (Sadenosylmethionine) to SAH (S-adenosylhomocysteine) in Abbreviations used: AR, androgen receptor; ARE, androgen response element; ChIP, chromatin immunoprecipitation; CS, charcoal-stripped; CT, cycle threshold; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GNMT, glycine N-methyltransferase; mTOR, mammalian target of rapamycin; NF-Y, nuclear factor-Y; PCa, prostate cancer; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B ( called Akt); RGA, reporter gene assay; RT–PCR, reverse transcription–PCR; SAH,

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