rs142362919 and rs7681187 Are cis‐Regulatory Variations for Human UGT2B28 in Breast

To the Editor: Glucuronidation is an important clearance pathway for numerous endobiotics and xenobiotics, including steroid hormones, bile acid, carcinogens, and clinical drugs and is catalyzed by the UDPglucuronosyltransferase (UGT) family.1–4 Among this family, UGT2B28 is an interesting member because of the presence of copy number variation.5 In the promoter region of this gene, 6 single nucleotide polymorphisms (SNPs), rs13139691 (–97 relative to the translation start), rs13124875 (–247), rs142362919 (–823), rs181302581 (–864), rs201366213 (–867), and rs7681187 (–1386), were identified with high minor allele frequency (>10%). 6 However, their role in UGT2B28 expression regulation has never been scrutinized. To resolve this issue, we amplified the promoter region by polymerase chain reaction with primers in Table S1 and, after digestion, cloned the segment into pGL3 basic vector (Promega, Madison, Wisconsin). After sequencing to determine the genotypes of these 6 sites, mutagenesis was performed by a Q5 Site-Directed Mutagenesis Kit (NEB, Ipswich, Massachusetts) with primer listed in Table S2 according to the manufacturer’s protocol. All plasmid constructs were transfected into human liver cell line HepG2 or breast line MCF-7, and luciferase activity was read by Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. In HepG2, the firefly luciferase read was rather close to negative control (pGL3-basic, result not shown), thus confirming that this gene is not expressed in liver.7 As far as breast tissue is concerned, mutagenesis at –1386 (A allele) and –823 (A allele) positions presented 29.3% and 34.2% increases in relative luciferase activity (P = .011 and <.01, Figure 1a), respectively, when compared with the original construct; plasmids with the other 4 mutageneses failed to alter promoter activity significantly (P > .05), which indicated that rs142362919 and rs7681187 are cis-regulatory variations for human UGT2B28 in breast. To further substantiate this, we also transfected the original construct and the plasmids from mutagenesis at –1386 and –823 into MDA-MB231, a human breast cancer cell line, and the result was displayed in Figure S1. The A allele at –823 also presented a significant increase (P< .05), but –1386 did not. The reason for the discrepancy between the 2 cell lines was unknown. We hypothesized that some factors might inhibit or compensate the effect of rs7681187 in MDA-MB-231, which might reflect the complexity of gene expression regulation in cancer cells. Because rs142362919 and rs7681187 were within the promoter region, it was reasonable to hypothesize that they lie within transcription factor binding sites and that they modify transcription factor binding affinity. We used Match at the TRANSFAC database (http://www.gene-regulation.com/cgi-bin/pub/program s/match/bin/match.cgi) to predict binding sites for potential transcription factors and found that – 1386 and –823 reside within a canonical POU class 2 homeobox 1 (POU2F1) and cutlike homeobox 1 (CUX1) or paired box 6 (PAX6) binding site, respectively. To verify this hypothesis, we performed chromatin immunoprecipitation by using EZ ChIP Kit (Upstate, USA) and antibody (Santa Cruz Biotechnology, Dallas, Texas) according to the manufacturer’s protocol. The relative enrichment was