Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

Abstract

The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase

“…Additionally, we place the hTERT transcriptional start site (TSS3) 64 bp upstream of the hTERT start codon. Several other transcriptional start points have been defined for hTERT and it is not uncommon for weak, TATA-less sequences such as the hTERT promoter to show complex transcriptional initiation patterns (Clark et al, 1998;Rudge and Johnson, 1999;Dong et al, 2000). The (Table 2).…”

miR-192/miR-215 Influence 5-Fluorouracil Resistance through Cell Cycle-Mediated Mechanisms Complementary to Its Post-transcriptional Thymidilate Synthase Regulation

“…One of the most studied mechanisms of TYMS expression variability are functional polymorphisms in the TYMS gene (15). Moreover, it is now well established that regulatory events at the level of gene amplification (16,17), transcription (18)(19)(20), posttranscription, translation, and posttranslation (21) are also involved in TYMS expression control (22). One example of TYMS posttranscriptional regulation is the rapid increase in TYMS enzyme activity described in various in vitro (23)(24)(25), in vivo, and clinical model systems following short-term exposure to fluoropyrimidines (26,27).…”

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

“…In human cells, two E2F consensus binding sites are found in the inverted repeat of the TS promoter. However, point mutation of neither of these sites affected TS gene expression (Dong et al, 2000;Lee and Johnson, 2000). Although studies of human tumour samples have examined the correlation between E2F levels and TS levels, no consistent picture has emerged.…”