Characterization of a hormone response element in the mouse N-acetyltransferase 2 (Nat2*) promoter

Abstract

Multiple variant alleles of the human arylamine N-acetyltransferase genes, NAT1* and NAT2*, alter the capacity of individuals to metabolize arylamines by N-acetylation. Although biochemical and genetic studies have improved our understanding of the molecular basis of the acetylation polymorphism in humans and other mammals, regulation of NAT* gene expression is not understood. In the present study, a segment of the 5'-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites located 112 to 151 bases upstream of the translational start site. Computer sequence analysis revealed a promoter-like region located within the region 530 bases upstream of the translational start site consisting of TATA boxes, upstream promoter elements such as a CAAT box and Sp1 binding site, regulatory elements such as a palindromic hormone response element (HRE), and enhancer regions such as an AP-1 transcription factor binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-palindromic HRE demonstrated positive regulation of the HSV-thymidine kinase 1 (tk1) promoter and induced the expression of chloramphenicol acetyltransferase (CAT). This induction was initiated by the addition of hormones such as 5alpha-dihydrotestosterone (DHT) or dexamethasone and was entirely dependent on the presence of androgen or glucocorticoid receptors, respectively. Together with recent discoveries regarding the effects of testosterone on the expression of Nat2* in mouse kidney during development, the findings reported in this article suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.

Figures

FIG. 1

Position and sequence of oligonucleotide primers. Primers Nat2-A and Nat2-B were employed in the primer extension analysis of mouse Nat2*. The gene structure of Nat2* is as depicted by Martell et al. (19). The 5′-flanking region (open bar), 5′- and 3′-untranslated regions (hatched bars), and coding exon (dotted bar).

FIG. 2

Determination of the transcription start sites of mouse Nat2* by primer extension assay. 32P-labeled primer Nat2-A (A) and primer Nat2-B (B) were hybridized with 150 μg of total RNA from C57BL/6J mouse liver (lane 1) and kidney (lane 2). Lanes a, c, g, and t are the reference dideoxy-sequencing reactions with each corresponding primer. Extended fragments are indicated by asterisks (*).

FIG. 3

Determination of the transcription start sites of mouse Nat2* by ribonuclease protection assay. Total RNA (85 μg) from B6.A mouse liver (lane 2), 65 μg kidney (lane 3), or 50 μg yeast (lane 4) was hybridized with 171-bp cRNA probe (lane 5). After treatment with RNase A/T, the protected fragments were analyzed on a 6% polyacrylamide/8 M urea gel. Sizes of the protected fragments were determined by comparison to the sequencing reaction on lane a, c, g, and t and to RNA markers in lane 1 (95 bp). The scheme (bottom) depicts the location of the probe (probe A) relative to the coding region (hatched bar) and 5′- and 3′-flanking regions (open bars) of mouse Nat2*.

FIG. 4

Nucleotide sequence of the 5′-flanking and untranslated regions of C57BL/6J mouse Nat2*. The sequence was determined for genomic clone 5H, previously isolated and mapped by Martell et al. (19). Translation start site (ATG) is indicated by a double arrow (+1) and the identified transcription initiation sites are indicated by asterisks (*). TATA boxes (underlined) and response elements (boxes) were identified as described in Materials and Methods. HRE, hormone response element; AP-1, activator protein 1 binding site; CCAAT Box and Sp1, upstream promoter elements. Also shown are the locations of primers Nat2-A and Nat2-B employed in primer extension analysis.

FIG. 5

Diagram constructs of CAT reporter genes containing multiple copies of hormone response elements (HRE). A 25-bp double-stranded oligonucleotide corresponding to the −579 to −560 nucleotide relative to the mouse Nat2* translation start site (+l), and flanked by BamHI sites, was synthesized and inserted into pUTKAT3 vector as one and two copies, as shown (dotted bars), upstream of the HSV-tk1 promoter element fused to the bacterial chloramphenicol acetyltransferase gene (open bars). The positive control construct (pG3tkCATF-3×HRE3) contains three HRE copies of the mouse Slp gene whereas the negative control (0 × HRE) has no copies of the HRE.

FIG. 6

The mouse Nat2* HRE displays positive gene regulation upon androgen stimulation. CV1 cells were cotransfected with 100 ng of mouse androgen receptor expression vector and 2 μg of the indicated CAT reporter construct (0× HRE, 1×HRE, 2 × HRE, or 3 × HRE3) shown in Fig. 5. After transfection, cells were incubated for 72 h with fresh media containing (A) no hormone or 340 nM of dihydrotestosterone (DHT), (B) no hormone or 100 nM dexamethasone (Dex), 340 nM DHT, or 100 nM of β-estradiol as indicated. (C) Cells were also transfected with 2×HRE in the presence or absence of mAR and incubated with or without DHT. Cells were harvested and heat-treated extracts were subjected to the nonchromatographic CAT assay described in Materials and Methods. CAT activities were normalized to total amount of protein (mg) and are expressed as average (dpm) of triplicate samples ± SEM.