A bioinformatics-based functional analysis shows that the specifically androgen-regulated gene SARG contains an active direct repeat androgen response element in the first intron.
We characterized the specifically androgen-regulated gene (SARG), which is expressed in the androgen receptor (AR) and glucocorticoid receptor (GR) positive cell line lymph node carcinoma of the prostate-1F5 (LNCaP-1F5). SARG mRNA expression can be up-regulated by androgens, but not by glucocorticoids. SARG mRNA expression is high in prostate tissue. SARG is composed of four exons and spans a region of 14.5 kbp on chromosome 1q32.2. Transcripts of 5.5, 3.3 and 2.3 kb are the result of alternative polyadenylation. SARG mRNA splice variants lack exon 2 and vary in length of exon 1. The SARG protein has a length of 601 amino acids and is located in the cytoplasm. By screening the 18 kbp genomic sequence flanking the transcription start site we identified the imperfect direct repeat 5'-TGTGCTaacTGTTCT-3'in intron 1 as an active androgen response element (ARE-SARG+4.6). A 569 bp genomic DNA fragment containing this element functioned as an androgen-specific enhancer in transiently transfected LNCaP-1F5 cells. ARE-SARG+4.6 cooperated with flanking sequences for optimal activity. Inactivation of ARE-SARG+4.6 completely abolished the androgen response of the enhancer. Chromatin immunoprecipitation (ChIP) experiments showed chromatin structural changes of the enhancer in the presence of R1881. ARE-SARG+4.6 was able to bind to the androgen receptor, but not to the glucocorticoid receptor, correlating with its androgen-specific activity in transfections.
Steketee
Karine
K
Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Ziel-van der Made
Angelique C J
AC
van der Korput
Hetty A G M
HA
Houtsmuller
Adriaan B
AB
Trapman
Jan
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics; D002878:Chromosomes, Human, Pair 1; D019295:Computational Biology / Q000379:methods; D004742:Enhancer Elements, Genetic / Q000235:genetics; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D026723:Polyadenylation; D011471:Prostatic Neoplasms / Q000235:genetics; D011944:Receptors, Androgen / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000728:Androgens; C499826:C1orf116 protein, human; D009687:Nuclear Proteins; D011944:Receptors, Androgen; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors