Characterization of the androgen receptor transcription unit

Abstract

ln this study the androgen receptor (AR) transcription unit is presented. Chapter II describes the isolation and characterization of one genomic clone, from which the amino acid sequence of the N-terminai domain of the hAR was deduced. This amino acid sequence was characterized by the presence of several homopolymeric stretches, of which a long a-stretch {20 residues) and a long G-stretch (16 residues) are most conspicuous. In combination with a previously described eDNA clone, which encoded the hAR DBD and LSD, an open reading frame of 2730 bp was deduced, encoding a protein of 910 amino acids. Next, the complete coding region of the hAR gene was isolated from a genomic library, as described in Chapter Ill. The information for the hAR was found to be separated over eight exons. The total lenght of the single copy hAR gene, which is located on the X-chromosome, exceeds 90 kb. As described in Chapter ll, the N-terminal domain is present in one single exon. The DBD is encoded in the exons 2 and 3, with each exon containing the information for one "zinc-finger". The LBD is encoded by the exons 4 to 8. Interestingly, the positions of the introns were found to be conserved between the hAR gene and the cPR and hER genes. The experiments described in Chapter IV extend the work of the Chapters II and Ill and deal with the characterization of the complete hAR eDNA and gene. Northern blot analysis of hAR mRNA identified two hAR mRNA species of 11 and 8 kb, respectively, in the human prostatic tumor cell line LNCaP. A full-length hAR eDNA was constructed from eDNA and genomic clones. Structurally, the 11 kb eDNA consists of a long 5'-UTR (1.1 kb), a 2.7 kb ORF, and a very long 3'·UTR (6.8 kb). The complete 5'-UTR and 3'-UTR were found to be encoded in the exons 1 and 8 of the hAR gene, respectively, fixing the number of exons in this gene at 8. The promoter of the hAR gene, located 1.1 kb upstream from the ATG translation initiation codon in exon 1, was structurally and functionally characterized. Two major sites of transcription initiation in a 13 bp region were identified by 51-nuclease protection experiments. DNA fragments, spanning these sites of initiation, conferred promoter activity upon a promoterless reporter gene construct. 51-nuclease protection experiments with RNA from COS cells, transiently transfected with these constructs, showed usage of the correct initiation sites. Structurally, the promoter lacks TATA/CCAAT boxes and potential regulatory elements consist of a short GC-box (-59/-321. which includes an Sp1 binding sequence (-46/-37), and a long homopurine stretch (-60/-117). The 3'-UTR contains two equally effective polyadenylation signals at a mutual distance of 221 bp. In addition, it was shown that the 8 kb hAR mRNA results from an alternative splice in the 3'-UTR, which does not affect the hAR OR