tert promoter sequences  (GenScript corporation)

Journal: Oncotarget

Article Title: Functional dissection of breast cancer risk-associated TERT promoter variants

doi: 10.18632/oncotarget.18226

Figure Lengend Snippet: TERT promoter variants with predicted change of regulatory motifs

Article Snippet: Wildtype and variant haplotype TERT promoter sequences (3915bp) harboring all major or all risk-associated alleles of rs2736107, rs2736108, rs145544133, rs2736109, rs3215401 and rs2853669 were synthesized by GenScript and were cloned into a pGL3 reporter vector (Promega E1751).

Techniques:

Journal: Oncotarget

Article Title: Functional dissection of breast cancer risk-associated TERT promoter variants

doi: 10.18632/oncotarget.18226

Figure Lengend Snippet: Cell lines were transfected with TERT promoter reporter constructs carrying risk alleles of the six candidate variants as indicated in the graph. (A) Comparison of wildtype and risk-associated haplotypes as well as the partial haplotypes carrying risk-associated alleles of SNPs 1-3 (rs2736107, rs2736108, rs2736109) or SNPs 4-6 (rs145544133, rs3215401, rs2853669), respectively. (B) Comparison of wildtype with single variant haplotypes for SNP4 (rs145544133), SNP5 (rs3215401), or SNP6 (rs3215401), and the partial SNP 4-6 (rs145544133, rs3215401, rs2853669) haplotype. Values were normalized to wildtype. The figures represent the estimated marginal effect (with 95% confidence interval) of data from at least 3 experiments performed on separate days. To account for the average difference between separate days a two-way ANOVA was performed with experiment day as a block variable. P-values from post-hoc comparisons between WT and the other groups were adjusted using Dunnett's correction (* P<0.05, ** P<0.01, ***P<0.001 ****P<0.0001). All statistical analyses were performed in log-scale, values were back-transformed for plots presented. An empty pGL3Basic vector was transfected into cells as negative control showing that TERT promoter activity is above background .

Article Snippet: Wildtype and variant haplotype TERT promoter sequences (3915bp) harboring all major or all risk-associated alleles of rs2736107, rs2736108, rs145544133, rs2736109, rs3215401 and rs2853669 were synthesized by GenScript and were cloned into a pGL3 reporter vector (Promega E1751).

Techniques: Transfection, Construct, Comparison, Variant Assay, Blocking Assay, Transformation Assay, Plasmid Preparation, Negative Control, Activity Assay

Journal: Oncotarget

Article Title: Functional dissection of breast cancer risk-associated TERT promoter variants

doi: 10.18632/oncotarget.18226

Figure Lengend Snippet: (A) Schematic location of SNP6 (rs2853669, major allele T/risk-associated allele C) in the TERT promoter with 3 major transcription factor binding sites: E2F, ETS and E-box. (B, C, D) ER-negative (ER-) MDA-MB 231 cells were transfected with siRNA then 24h later transfected with TERT promoter constructs. The wildtype construct was compared to the construct carrying the risk-associated allele of SNP6 (rs2853669) and the risk-associated haplotype after treatment with control siRNA or MYC targeting siRNA (B) , ETS2 targeting siRNA (C) or MYC and ETS2 targeting siRNA (D) . Asterisks next to the legend illustrate the significance of the overall interaction between control siRNA and MYC and/or ETS2 targeting siRNA. Similar data were obtained for ER-positive (ER+) MCF-7 cells and Bre80 normal breast cells . (E) Knock-down efficiency was determined by western blot analysis and was quantified for one experiment by ImageJ (western blots are shown in ). (F) Position of SNP1 (rs2736107) in the estrogen responsive element of the TERT promoter. (G) ER+ MCF-7 cells were cultured in phenol-red free media and treated with 10nM fulvestrant for 48h. Afterwards, cells were transfected with TERT promoter reporter constructs carrying major (wildtype) alleles or risk-alleles as indicated. The medium contained 20nM estrogen or DMSO as a control during and after transfection. The wildtype construct was compared to that carrying the risk-associated allele of SNP1 (rs2736107) alone, the partial haplotypes carrying risk-associated alleles of SNPs 1-3 (rs2736107, rs2736108, rs2736109) or SNPs 4-6 (rs145544133, rs3215401, rs2853669), respectively, and the risk-associated haplotype carrying all six risk-associated alleles. (H) Estrogen induction was verified by pGL3 vector containing an estrogen responsible element. Data are shown for at least three experiments (B, C, G) or two experiments (D) . Three-way ANOVA was used to assess the effect of group, estrogen/siRNA and the interaction of group and estrogen/siRNA. Experiments were performed on separate days and results were combined by including day as a blocking factor into the ANOVA. The figures represent the estimated marginal effect (with 95% confidence interval) of each treatment combination after accounting for the average difference between separate days. Multiple comparisons between groups of interest were defined via contrasts. P-values were adjusted using Bonferroni's multiple hypotheses testing adjustment (** P<0.01, ***P<0.001 ****P<0.0001). All statistical analysis was performed in log-scale.

Article Snippet: Wildtype and variant haplotype TERT promoter sequences (3915bp) harboring all major or all risk-associated alleles of rs2736107, rs2736108, rs145544133, rs2736109, rs3215401 and rs2853669 were synthesized by GenScript and were cloned into a pGL3 reporter vector (Promega E1751).

Techniques: Binding Assay, Transfection, Construct, Control, Knockdown, Western Blot, Cell Culture, Plasmid Preparation, Blocking Assay

Journal: Oncotarget

Article Title: Functional dissection of breast cancer risk-associated TERT promoter variants

doi: 10.18632/oncotarget.18226

Figure Lengend Snippet: The six promoter SNPs that have been analyzed (in green with rs-number) and the SNP rs2736098 (not evaluated in this study) in the second exon of the TERT gene (in grey) are shown. They reside in one LD block and are part of breast cancer risk association signal 1. rs2736107 (SNP1) resides in an estrogen responsive element (ERE). The risk-associated alleles of SNP rs2736108 (SNP2) and rs2736109 (SNP3) were associated with open chromatin, while rs145533144 (SNP4) had no effect in any of the conducted analysis. rs3215401 (SNP5) reduced TERT promoter activity in breast cancer cell lines but the mechanism remains unclear. rs2853669 (SNP6) reduced TERT promoter activity in a similar manner. This polymorphism resides in an ETS binding site next to an E-box motif for c-MYC binding and ChIP experiments indicated an allele-specific effect of rs2853669 (SNP6) on GABPA binding, a transcription factor of the ETS family. An interaction of GABPA with c-MYC or other ETS family members at this position at the TERT promoter is unknown. The somatic mutations (orange stars) found in several cancers at position −124 and −149 create novel ETS binding sites and have been found to increase TERT transcription by GABPA binding in other cell lines, which activates a switch for chromatin folding.

Article Snippet: Wildtype and variant haplotype TERT promoter sequences (3915bp) harboring all major or all risk-associated alleles of rs2736107, rs2736108, rs145544133, rs2736109, rs3215401 and rs2853669 were synthesized by GenScript and were cloned into a pGL3 reporter vector (Promega E1751).

Techniques: Blocking Assay, Activity Assay, Binding Assay