Genome-wide association studies (GWASs) have revealed SNP rs889312 on 5q11. = 0.87C0.93, pcond = 1.4? 10?4). Twenty-six percent of the prioritized candidate variants coincided with four putative regulatory elements that interact with the promoter through chromatin looping and impact promoter activity. Functional analysis indicated that this malignancy risk alleles of four candidates (rs74345699 and rs62355900 [iCHAV1], rs16886397 [iCHAV2a], and rs17432750 [iCHAV3]) increased transcriptional activity. Chromatin immunoprecipitation analysis revealed diminished GATA3 binding to the minor (cancer-protective) allele of rs17432750, indicating a mechanism for its action. We propose that the malignancy risk alleles take action to increase expression in?vivo and might promote breast malignancy cell survival. Introduction One of the first genome-wide association studies (GWASs) for breast malignancy (MIM 114480) susceptibility recognized a 5q11.2 SNP (rs889312) associated with risk of breast cancer in women of European ancestry.1 In the most recent analyses by the Floxuridine IC50 Breast Malignancy Association Consortium (BCAC), the minor allele of rs889312 was associated with a per-allele odds ratio (OR) = 1.12 (95% confidence interval [CI] = 1.10C1.15; ptrend = 1.8? 10?26).2 The association was stronger for estrogen-receptor-positive (ER+) disease (OR = 1.14, 95% CI = 1.11C1.17, p = 1.1? 10?26 in the most recent BCAC analysis) but was also seen for estrogen-receptor-negative (ER?) disease (OR = 1.06, 95% CI?= 1.03C1.10, p = 0.0024) and triple negative disease (OR = 1.11, 95% CI = 1.02C1.20, p = 0.016).3 SNP rs889312 was also reported to be associated with an increased breast cancer risk in carriers of (MIM 600185) mutations.4 The GWAS SNP rs889312 lies approximately 80 kb centromeric to (MIM 600982), the gene encoding mitogen-activated protein kinase kinase kinase 1, also known as MEK kinase 1 (MEKK1), a stress-induced serine/threonine kinase with apparent dual functions: MEKK1 induces cell proliferation through a RAS-RAFin breast cancer pathogenesis: driver mutations have been observed in luminal A and B type breast tumors,11 and expression has been associated with specific breast tumor subtypes.12 In this study, we performed genetic epidemiological analyses on all common variants at 5q11.2, together with in?silico and in?vitro analyses of candidate causal variants, and identified strong candidates that we propose are functionally related to breast malignancy risk. Specifically, we provide evidence that these associations are mediated through promoter and the transcription start site (chr5: 56,109,070C56,110,997, GRch37) into the MluI and HindIII sites of pGL3-Basic. To assist cloning, AgeI and SbfI sites were inserted into the BamHI and SalI sites downstream of the luciferase gene. A 1,575?bp putative regulatory element (PRE)-A fragment, a 1,765?bp PRE-B2 fragment, a 2,357?bp PRE-B3 fragment, a 2,203?bp PRE-C fragment, and a 1,519?bp PRE-D fragment were generated by PCR using primers designed with AgeI and SbfI sites and cloned into the modified pGL3-promoter construct. PRE-B was too large (7 kb) to be cloned in its entirety, so three subregions termed PRE-B1, PRE-B2, and PRE-B3 were cloned separately. The minor alleles of individual SNPs were launched into promoter and PRE sequences, made up of the major alleles of any other causal candidate variants, by overlap extension PCR. Sequencing of all constructs confirmed variant incorporation (AGRF, Brisbane). PCR primers are outlined in Table S2. For the PRE-B1 construct, a 2,129?bp region spanning chr5: 56,028,968C56,031,097 (GRCh37) was synthesized with AgeI and SbfI sites incorporated at the 5 and 3 ends (GenScript, Floxuridine IC50 Piscataway) to assist cloning into the promoter construct. The cloned regions are highlighted in Physique?2B. Physique?2 Candidate Causal Variants Are Located in PREs that Interact with the Promoter Reporter Assays and Estrogen Induction Bre-80 and MCF7 cells were transfected with equimolar amounts of luciferase reporter plasmids and 50?ng of pRLTK transfection control plasmid Floxuridine IC50 with Lipofectamine 2000. The total amount of transfected DNA was kept constant at 600?ng for each construct by the addition of pUC19 as a carrier plasmid. Luciferase activity was measured 24?hr posttransfection by the Dual-Glo Luciferase Assay System. Normalizing firefly luciferase activity to luciferase corrected for differences in transfection efficiency or cell-lysate preparation. For the assays under basal conditions, the activity of each construct was calculated in relation to the activity (defined as 1) of the construct made up of T the promoter alone. For estrogen-induction assays, we treated cells as explained.18 In brief, 24?hr after plating MCF7 cells into wells, we replaced medium with that containing 10?nM fulvestrant for 48? hr to inhibit estrogen-induced gene expression and thereby produce a baseline of expression for reporter assays. Cells were then incubated with new medium made up of either 10?nM estrogen (17-estradiol) or DMSO (vehicle control) and transfected with reporter plasmids. Luciferase assays were performed as above after 24?hr. Statistical significance was.