SMUG1 is a recently discovered uracil-DNA glycosylase with the ability to remove uracil from single-stranded as well as double-stranded DNA. SMUG1 also has the capacity to excise oxidized pyrimidine bases such as 5-hydroxymethyluracil and 5-formyluracil from DNA. Very little is known about the regulation of this enzyme. Therefore, we undertook this study to begin to elucidate the mechanisms of hSMUG1 gene expression. Northern blot analysis performed on mRNAs derived from different cell lines reveals that the steady-state levels of hSMUG1 transcript are about 10-fold lower relative to UDG. In addition to the 1.6kb transcript known to encode a functional hSMUG1 protein, an alternate 0.7kb transcript was uncovered that contains an open reading frame. Interestingly, this alternate transcript is missing a carboxy-terminal domain which is necessary for catalytic activity. Utilizing a luciferase reporter assay system we show that significant promoter activity is associated with a 2000bp region, located immediately upstream of the first transcribed, non-translated exon. 5′ deletion analysis of this 2000bp region reveals that there are both negative and positive regulatory elements that control expression of SMUG1. Using electrophoretic mobility shift analysis we show that a number of DNA-protein complexes are formed within the region (-705 to -604) of positive regulation. At least two of these complexes contain the transcription factor NFI/CTF as demonstrated by oligonucleotide competition studies with NFI/CTF consensus sequence containing both protein-binding half-sites. We further demonstrate that purified NFI-C protein will bind to this positive regulatory region within the hSMUG1 gene. DNase I footprint analysis reveals that the 3′ half-site is protected when using crude nuclear extract as a protein source. However, the introduction of mutations into either or both of the half-sites indicates that the individual half-sites contribute to NFI/CTF binding. Overexpression of NFI-C in NIH-3T3 cells results in an increase in SMUG1 enzyme activity. Collectively, these data indicate that the NFI/CTF consensus site may function as a cis-element in the SMUG1 promoter and that this transcription factor contributes to the positive regulation of SMUG1 gene expression.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology