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, ␤ (nucleotides ؊189 to ؊167), ␣ (؊98 to ؊76), Inr (؊4 to ؉16), and ␥ (؉34 to ؉61), together regulating promoter activity. The nearly identical upstream elements, ␣ and ␤, 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 ␣ element as a target. Transient transfection assays demonstrate that the ␣ and ␤ elements are mainly important for the R1 promoter strength and suggest that YY1 functions as an activator.The enzyme ribonucleotide reductase is essential for de novo synthesis of deoxyribonucleotides. In this perspective, ribonucleotide reductase plays a central role in providing the precursors for DNA synthesis. Mammalian ribonucleotide reductase consists of two non-identical homodimeric subunits, proteins R1 and R2 (1). Protein R1 (large subunit) contains the active site and allosteric sites for regulating enzyme activity. Protein R2 (small subunit) carries a tyrosyl free radical essential for enzyme activity. In quiescent or differentiated cells, none of the two subunits can be detected. In proliferating cells, the expression of protein R2 is S phase specific, whereas protein R1 shows a constant level throughout the cell cycle (2-4). However, both the R1 and R2 mRNA levels vary in parallel during the cell cycle with negligible levels during G 0 /G 1 and maximal levels during S phase (5, 6). The R1 and R2 promoters are also activated in resting cells upon UV irradiation leading to nucleotide excision repair (7).Genomic clones covering the mouse R1 and R2 genes have been isolated (8, 9). The R2 promoter contains a TATA-box and a proximal element, called ␣, that contains an NF-Y binding CCAAT motif (10).The R1 gene consists of 19 exons that covers 26 kb. 1 In contrast to the R2 promoter, the R1 promoter is TATA-less. DNase I footprinting assays proximal to the transcription start revealed two protected regions, ␣ (nt Ϫ98 to Ϫ76) and ␤ (nt Ϫ189 to Ϫ167) (8). These two regions are each 23 nt and identical except for one nucleotide. Three DNA-protein complexes, A, B, and C, are formed when oligonucleotides corresponding to the ␣ or ␤ footprints are used in gel shift assays. Complexes A and B are present at constant levels during the cell cycle, whereas c...

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“…YY1 was reported to control several S-phase-induced genes (Johansson et al 1998;Wu and Lee 2001). Overexpression of YY1 was reported to induce DNA synthesis (Petkova et al 2001).…”

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