Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites

Abstract

The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

Cyclic AMP regulates expression of the RIα subunit of cAMP‐dependent protein kinase through an alternatively spliced 5′ UTR

“…Mutation of this element in P-glycoprotein promoter constructs reduced expression of reporter protein by 50% in P-glycoprotein overexpressing cells [37]. In contrast, no effect of MED-1 mutation was shown in the mouse thymidylate synthase promoter [38]. According to these contradicting results, the function of this element still remains unclear.…”

Modulation of the typical multidrug resistance phenotype by targeting the MED-1 region of human MDR1 promoter

“…However, a recent report states that inactivation of the MED-1 element in TATA-less and initiatorless mouse thymidylate synthase promoter has no effect on promoter strength and is not an essential component of TATA-less promoters with complex transcriptional inhibition patterns. 45 The human MED-1 element found in the MDR1 gene represents a particular case for different reasons: we have mentioned its upstream location (93 bp from the initiator window) and its inverted sequence compared with the consensus one; moreover, the decoy we used (ds-ODN3) covers the MED-1 sequence and does not seem to interfere with the CAAT box (which is located 7 bp upstream), particularly since no effect was noticed in CEM/s cells. In addition, inactivation of MED-1 in MDR1 genes resulted in an important decrease of promoter strength.…”

“…A synthetic cellular model (NIH-EGFP) and highly resistant human CEM/VLB0. 45 tion initiation start. 11 A new class of RNA polymerase II functioning with TATA-less promoters has recently been described; such promoters share the same arrangement of multiple transcription start points inside the transcription window.…”

The RNA Polymerase II Core Promoter

“…Third, the multiple start site downstream element (MED-1) was identified in TATA-less promoters that have unclustered, multiple start sites (195). The MED-1 element was observed to contribute to transcriptional activity in two of three promoters tested (195)(196)(197). In the future, it will useful to study these elements further as well as to identify additional core promoter motifs.…”