A novel single nucleotide polymorphism within the 5' tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity.
Thymidylate synthase (TS) gene expression is modulated by a polymorphism in the 5' regulatory region of the gene. The polymorphism consists mainly of either two repeats (2R) or three repeats (3R) of a 28-bp sequence, yielding greater TS gene expression and protein levels with a 3R genotype. Two USF family E-box consensus elements are found within the tandem repeats of the 3R genotype, and one is found within the 2R genotype. These elements bind USF proteins in vitro by electrophoretic mobility shift analysis and in vivo by chromatin immunoprecipitation assay. We show that the additional USF consensus element within the 3R construct confers greater transcriptional activity relative to the 2R construct. Mutagenesis of the USF sites shows that the transcriptional regulation of TS is dependent, in part, on USF proteins binding within the tandem repeats. In addition, we identified a novel G-->C single nucleotide polymorphism in the second repeat of 3R alleles within the USF consensus element that alters the ability of USF proteins to bind and thus alters the transcriptional activation of TS gene constructs bearing this genotype. Through RFLP analysis, we determined the respective frequencies of the C allele (3RC) among all 3R alleles in non-Hispanic whites, Hispanic whites, African Americans, and Singapore Chinese to be 56%, 47%, 28%, and 37%, respectively. Based on our findings, this novel single nucleotide polymorphism should be considered when the 5' tandem repeat polymorphism is being used as a predictor of clinical outcome to TS inhibitors.
Mandola
Michael V
MV
Department of Molecular Biology, The University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084, USA.
Stoehlmacher
Jan
J
Muller-Weeks
Susan
S
Cesarone
Gregory
G
Yu
Mimi C
MC
Lenz
Heinz-Josef
HJ
Ladner
Robert D
RD
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins; D024721:E-Box Elements; D005006:Ethnicity / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; D008156:Luciferases; D013940:Thymidylate Synthase