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.
Chabes
Anna Lena
AL
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
Björklund
Stefan
S
Thelander
Lars
L
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050704:E2F4 Transcription Factor; D016193:G1 Phase; D016195:G2 Phase; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006168:Guinea Pigs; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012264:Ribonucleotide Reductases / Q000096:biosynthesis* / Q000235:genetics; D016196:S Phase; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; C493754:E2f1 protein, mouse; C493763:E2f4 protein, mouse; D014157:Transcription Factors; D008156:Luciferases; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D003850:Deoxyribonuclease I