Two YY-1-binding proximal elements regulate the promoter strength of the TATA-less mouse ribonucleotide reductase R1 gene

Abstract

Ribonucleotide reductase is essential for DNA synthesis. In mammalian cells, the enzyme consists of two nonidentical subunits, proteins R1 and R2. The expression of the mouse R1 and R2 genes is strictly correlated to S phase. Using promoter-reporter gene constructs, we have defined a region of the TATA-less mouse ribonucleotide reductase R1 gene promoter that correlates reporter gene expression to S phase. This is demonstrated in stably transformed cells both synchronized by serum starvation and separated by centrifugal elutriation, suggesting that the R1 gene expression during the cell cycle is mainly regulated at the transcriptional level. The region contains four protein-binding DNA elements, β (nucleotides -189 to -167), α (-98 to -76), Inr (-4 to +16), and γ (+34 to +61), together regulating promoter activity. The nearly identical upstream elements, α and β, each form three DNA-protein complexes in gel shift assays. We have identified YY1 as a component in at least one of the complexes using supershift antibodies and a yeast one-hybrid screening of a mouse cDNA library using the α element as a target. Transient transfection assays demonstrate that the α and β elements are mainly important for the R1 promoter strength and suggest that YY1 functions as an activator.