A Potential H-DNA Element in the MUC1 Promoter Does Not Influence Transcription

Gurcharan S Pahwa,L James Maher,Michael A Hollingsworth

doi:10.1074/jbc.271.43.26543

Gurcharan S Pahwa, L James Maher + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.43.26543

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Abstract

A purine/pyrimidine mirror repeat element (M-PMR3) in the MUC1 promoter has been shown to form H-DNA under in vitro conditions. We investigated this element for biological function in the regulation of transcription of this gene. Chloramphenicol acetyltransferase reporter-promoter constructs were prepared in which the mirror repeat element (PMR3) was intact, deleted, or modified, and their activities were evaluated by transient transfection assays into the cell lines Capan-2, PANC1, and HT-29. Deletion or modification of M-PMR3 increased expression of chloramphenicol acetyltransferase activity in MUC1-expressing cells; however, a role for an H-DNA structure in this activity was not supported by the results.

Highlights

Purine/pyrimidine mirror repeat elements (PMRs)1 in the 5Ј upstream region of the human CFTR and MUC1 genes are sensitive to S1 nuclease and chemical probes of single-stranded DNA character, consistent with the formation of H-DNA in vitro under conditions of acidic pH and plasmid supercoiling [1, 2]
The results suggest that mirror symmetry in the sequence and potential for H-DNA in the element do not influence transcriptional activity of this promoter in transient transfection assays
The CAT construct with 790 bp of upstream sequence was used in studies that evaluated the effect of deleting and modifying the M-PMR3 element

Summary

Introduction

Purine/pyrimidine mirror repeat elements (PMRs) in the 5Ј upstream region of the human CFTR and MUC1 genes are sensitive to S1 nuclease and chemical probes of single-stranded DNA character, consistent with the formation of H-DNA in vitro under conditions of acidic pH and plasmid supercoiling [1, 2]. A nuclear protein of approximately 27 kDa binds to the purine-rich strand of these elements but not to the pyrimidinerich strand or to double-stranded oligonucleotides of the same sequence [1]. The studies reported here were designed to determine if PMR elements in the MUC1 promoter play a role in regulating transcription of the MUC1 gene. The results suggest that mirror symmetry in the sequence and potential for H-DNA in the element do not influence transcriptional activity of this promoter in transient transfection assays

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Conclusion