PMC

SNP rs6893267 influences enhancer E activity. Luciferase activity of enhancer-Myc-luciferase constructs in prostate cell line LnCAP with or without β-catenin/Tcf-4 (25 ng of each plasmid). The G and T allele variants of enhancer E are indicated. Multiple repeats were performed for each allele with statistically significant P values of P < 0.0022 (Datasets S1–Dataset S4).

Enhancer reporter assay. (A) The enhancers were cloned in front of a Myc promoter-driven or TK promoter-driven luciferase-GFP reporter. (B) Luciferase activity of enhancer-Myc-luciferase constructs in prostate cell line LnCAP, PC3, and colorectal cell line SW620. (C) Luciferase activity of enhancer-TK-luciferase constructs in prostate cell line LnCAP, PC3, and colorectal cell line SW620. Enhancer reporter plasmids were cotransfected into cell lines as described in . Luciferase activity was measured 24 h after transfection by using the Dual-Glo Luciferae assay system.

Enhancer E forms close contact with Myc promoter in 3C assay. (A) The 3C primer locations. (B) PCR results in the presence or absence of β-catenin and/or Tcf-4 by using the indicated amounts of LnCAP BglII digested/ligated genomic DNA or nondigested/ligated genomic DNA (gDNA). The concentration of DNA templates for PCR amplifications was determined by UV absorbance and confirmed by qPCR. (C) Sequence of the recovered PCR product with primer locations and BglII site are indicated.

Enhancer E interacts with Tcf-4 in vivo. (A) β-catenin/Tcf-4 can stimulate enhancer E activity in the luciferase reporter assay. Luciferase activity of Myc-luciferase constructs (with and without enhancer E) in prostate cell line LnCAP with (+)/without (−) β-catenin and/or Tcf-4. (B) Tcf-4 binds to enhancer E in LnCAP cells in a ChIP assay. ChIP was performed (lanes 2, 3, 5, and 6) by using genomic LnCAP DNA, a Tcf-4 monoclonal antibody (Tcf-4), or total mouse IgG (IgG), and PCR primers directed against the Myc promoter (Myc) or enhancer E as indicated. Lanes 1 and 4 contain positive control PCR products generated from input genomic DNAs.

Computationally predicted enhancers in the 8q24 cancer-associated gene desert region. Enhancers were predicted by aligning the human 8q24 genomic sequence with dog and mouse orthologs by using EEL. A physical map of 8q24, including Myc and 800 kb enveloping the 8q24 cancer associated region, are shown. The three prostate cancer-associated regions not in LD are shown in orange. The three most significant SNPs (rs16901979, rs6983267, and rs1447295) are illustrated above the region. Enhancers predicted by Heintzman et al. () and Tcf-4 binding sequences identified by Hatzis et al. () are indicated. The top seven predicted enhancers (along with their relative scores) are indicated as A–G on the map. Enhancer E is shown in detail together with its three predicted Tcf-4 binding sites, conservation score, and regulatory potential score from UCSC genome database.