S phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region

Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) - 672 to - 527 from the transcription start site) and one proximal promoter repressive element ( located at nt - 112 to - 107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element ( nt - 79 to - 75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.