A Novel Nomenclature for Repeat Motifs in the Thymidylate Synthase Enhancer Region and Its Relevance for Pharmacogenetic Studies

Inhibition of thymidylate synthase (TS) is the primary mode of action for 5-fluorouracil (5FU) chemotherapy. TS expression is modulated by a variable number of tandem repeats in the TS enhancer region (TSER) located upstream of the TS gene (TYMS). Variability in the TSER has been suggested to contribute to 5FU-induced adverse events. However, the precise genetic associations remain largely undefined due to high polymorphism and ambiguity in defining genotypes. To assess toxicity associations, we sequenced the TSER in 629 cancer patients treated with 5FU. Of the 13 alleles identified, few could be unambiguously named using current TSER-nomenclature. We devised a concise and unambiguous systematic naming approach for TSER-alleles that encompasses all known variants. After applying this comprehensive naming system to our data, we demonstrated that the number of upstream stimulatory factor (USF1-)binding sites in the TSER was significantly associated with gastrointestinal toxicity in 5FU treatment.

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Genetic Heterogeneity of Variable Number Tandem Repeats in Thymidylate Synthase Gene in Colorectal Cancer Patients

The International Journal of Biological Markers ◽

10.1177/172460080401900413 ◽

2004 ◽

Vol 19 (4) ◽

pp. 332-336 ◽

Cited By ~ 4

Author(s):

M. Agostini ◽

S. Pucciarelli ◽

P. Calandra ◽

F. Villani ◽

M. Lise ◽

...

Keyword(s):

Thymidylate Synthase ◽

Tandem Repeats ◽

Colorectal Adenocarcinoma ◽

Variable Number ◽

Mismatch Repair Genes ◽

Pcr Product ◽

Group A ◽

Microsatellite Stability ◽

Group B ◽

Repair Genes

Purpose To analyze the genetic variability in a variable number of tandem repeats (VNTR) in the thymidylate synthase (TS) enhancer promoter region and assess the influence of functional alterations in mismatch repair genes by analyzing constitutional and tumoral DNA from patients with colorectal adenocarcinoma with a high microsatellite instability (MSI-H) or microsatellite stability (MSS) status. Patients and methods Patients who underwent surgery for colorectal adenocarcinoma were selected from the colorectal database of our institute and, on the basis of MSI status, assigned to a study group and a control group: group A, MSI-H; group B, MSS. Microsatellite status was investigated using the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, D17S250). In MSI-H patients an additional analysis was made of the microsatellite loci D18S61 and D18S58, both mapping in the region containing the TS gene (18p11.2–11.32). Based on the number of altered microsatellites (≥2, 1, or 0), tumors were considered as having high (MSI-H) or low (MSI-L) instability or microsatellite stability (MSS), respectively. Genotyping for thymidylate synthase promoter polymorphism was carried out on constitutional and tumor DNA of each patient by PCR amplification of the polymorphic region. Results MSI-H was found in 55 patients (group A) and MSS in 50 patients (group B). In none of the MSI-H patients was microsatellite instability found in the additional D18S61 and D18S58 loci. In five group A and ten group B cases the analysis was not performed because constitutional DNA and/or tumoral DNA were not amplifiable. Homozygotes for the triple repeat variant (3R/3R) displayed only the large PCR product, homozygotes for the double repeat variant (2R/2R) displayed only the smaller PCR product, while heterozygotes (2R/3R) displayed both the larger and smaller PCR products. In 3/50 (6%) group A patients and 5/40 (12%) group B patients repeat variations were found in tumoral DNA. Conclusion Our findings demonstrate that there is genetic homogeneity between constitutional and tumoral DNA but do not support the hypothesis that mismatch repair genes are involved in VNTR recombinant events in TS gene variability.

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Genetic heterogeneity of variable number tandem repeats in thymidylate synthase gene in colorectal cancer patients

The International Journal of Biological Markers ◽

10.5301/jbm.2008.3961 ◽

2004 ◽

Vol 19 (4) ◽

pp. 332-336 ◽

Cited By ~ 1

Author(s):

M. Agostini ◽

S. Pucciarelli ◽

P. Calandra ◽

F. Villani ◽

M. Lise ◽

...

Keyword(s):

Colorectal Cancer ◽

Cancer Patients ◽

Thymidylate Synthase ◽

Genetic Heterogeneity ◽

Tandem Repeats ◽

Variable Number ◽

Colorectal Cancer Patients ◽

Variable Number Tandem Repeats

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Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography

Analytical Biochemistry ◽

10.1016/j.ab.2004.07.025 ◽

2004 ◽

Vol 334 (2) ◽

pp. 276-283 ◽

Cited By ~ 3

Author(s):

Hany Ezzeldin ◽

Cornelia Hoffmayer ◽

Richie Soong ◽

Martin R. Johnson ◽

Adam Lee ◽

...

Keyword(s):

Thymidylate Synthase ◽

High Performance ◽

Allelic Imbalance ◽

Tandem Repeats ◽

Simultaneous Detection ◽

Variable Number ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Gene Enhancer ◽

Variable Number Tandem Repeats

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The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites.

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.4079 ◽

1989 ◽

Vol 9 (9) ◽

pp. 4079-4082 ◽

Cited By ~ 14

Author(s):

T Deng ◽

Y Li ◽

K Jolliff ◽

L F Johnson

Keyword(s):

Thymidylate Synthase ◽

Promoter Region ◽

Promoter Activity ◽

Initiation Site ◽

Essential Elements ◽

Site Directed Mutagenesis ◽

Upstream Stimulatory Factor ◽

Transcriptional Initiation ◽

Close Proximity ◽

Stimulatory Factor

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.

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Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression

Biochemical Journal ◽

10.1042/bj20021585 ◽

2003 ◽

Vol 371 (3) ◽

pp. 675-686 ◽

Cited By ~ 27

Author(s):

Cyrus C. MARTIN ◽

Christina A. SVITEK ◽

James K. OESER ◽

Eva HENDERSON ◽

Roland STEIN ◽

...

Keyword(s):

Gene Expression ◽

Binding Sites ◽

Fusion Gene ◽

Catalytic Subunit ◽

Related Protein ◽

Upstream Stimulatory Factor ◽

Factor Binding ◽

E Box ◽

Stimulatory Factor

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived βTC-3 cells revealed that multiple promoter regions, located between −306 and −97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans-acting factor-binding sites in the IGRP promoter that were identified in βTC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans-acting factor-binding sites located between −97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the −306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 neurogenic differentiation/β-cell E box transactivator 2 and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

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The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.4079-4082.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 4079-4082

Author(s):

T Deng ◽

Y Li ◽

K Jolliff ◽

L F Johnson

Keyword(s):

Thymidylate Synthase ◽

Promoter Region ◽

Promoter Activity ◽

Initiation Site ◽

Essential Elements ◽

Site Directed Mutagenesis ◽

Upstream Stimulatory Factor ◽

Transcriptional Initiation ◽

Close Proximity ◽

Stimulatory Factor

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Transcription factors acting on the promoter of the rat fatty acid synthase gene

Biochemical Society Transactions ◽

10.1042/bst0301070 ◽

2002 ◽

Vol 30 (6) ◽

pp. 1070-1072 ◽

Cited By ~ 24

Author(s):

M. Schweizer ◽

K. Roder ◽

L. Zhang ◽

S. S. Wolf

Keyword(s):

Transcription Factors ◽

Fatty Acid ◽

Binding Sites ◽

Fatty Acid Synthase ◽

Regulatory Element ◽

Upstream Stimulatory Factor ◽

Element Binding Protein ◽

Sterol Regulatory Element ◽

Responsive Element ◽

Stimulatory Factor

Fatty acid synthase (FAS), one of the main lipogenic enzymes, converts dietary calories into a storage form of energy. The transcription factors, stimulatory proteins 1 and 3 (Sp1 and Sp3), nuclear factor Y (NF-Y), upstream stimulatory factor (USF) and sterol regulatory element binding protein-1 (SREBP-1) have cognate binding sites on the promoter of the FAS gene. It was shown that Sp1 and NF-Y interact co-operatively at the diet-induced DNase I-hypersensitive site at position —500. Adjacent binding sites for NF-Y and Sp1 have also been found between —71 and —52, and —91 and —83. cAMP regulation is mediated via the inverted CAAT element (ICE) at —99 to —92, which binds NF-Y. The FAS insulin-responsive element 3 (FIRE3)-binding site at —71 to —52 is capable of binding NF-Y, USF and SREBP-1, and is required for the sterol response in conjunction with the co-activator NF-Y around —100. Surprisingly, both FIRE3 and ICE are also necessary for the response to retinoic acid that plays a role in development and is an essential component of the diet.

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