Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1

Abstract

Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by RT-PCR and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5'-untranslated region (5'-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5'-UTR exon present in P1 mRNA. CAP-dependent amplification of 5' P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatomaderived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435 bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk.

Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1

“…Many of the NAT1 haplotypes that have been identified in human populations contain a single SNP in the 870 bp NAT1 coding region (Table 1). Although the functional effects of SNPs in the promoter and other regions of the NAT1 gene may also affect phenotype, 22,23 our investigation focused on the functional effects of the SNPs identified in the NAT1 coding region.…”

Inflammatory cytokines suppress arylamine N -acetyltransferase 1 in cholangiocarcinoma cells

“…However, information regarding NAT1 expression is currently not available. Regulation of NAT1 expression has been under study for a long time [23,28] . Previous studies have shown that suppression of NAT1 activity by substrates or oxidant species is due to direct inhibition of the enzyme molecules [13,29] , since the expression of NAT1 mRNA does not show any alteration [29] .…”

Functional analysis of N-acetyltransferase (NAT1*14B and NAT1*10) in complete NATb and NATa mRNA.

“…However, the relative contribution of transcripts derived from the NATa promoter is not known. Transcripts derived from the NATb promoter have been found in all tissues studied but transcripts derived from the NATa promoter have been found in liver, kidney, lung, and trachea (Barker et aL, 2006;Husain et aL, 2004). It is possible that NATa transcripts are expressed in a wider range of tissues, but only when the cell is under specific environmental stress or in a disease state.…”

“…Less is known about the alternative, NATa promoter. The NATa promoter region has been mapped to a 435 bp region upstream of exon 1 (Barker et aL, 2006). The relative strength of the NATa promoter was low compared to the NATb promoter and pGL3-control (containing the strong SV40 promoter) in HepG2 cells (Barker et aL, 2006;Husain et aL, 2004).…”

“…Differences in translational efficiencies exist between transcripts originating at each promoter, but the biological importance of this remains unclear (Butcher et aL, 2005). Six different mRNAs have been identified to begin at the first promoter, NATa (also known as P3), which is located 50.1 kb upstream of the NAT1 ORF (Barker et aL, 2006). Five different mRNAs have been identified which begin at the second promoter, NA Tb (also known at P 1), located just upstream of exon 4 (11.8 kb of the NAT1 ORF) (Butcher et aL, 2005;Husain et aL, 2004) (Figure 1 -4).…”