Abstract
In recent years it has become apparent that the cellular machinery governing cell cycle progression and transcription control are often homologous in yeast and mammalian cells. We and others have previously shown that the SP family of mammalian transcription factors regulates the transcription of a number of genes whose activities are governed by the product of the retinoblastoma (Rb) susceptibility gene, including c-FOS, c-MYC, TGFbeta-1, IGF-II, and c-JUN. To determine whether a similar pathway of transcriptional regulation may function in yeast, we explored the possibility that transcription factors with nucleotide-binding specificities akin to those of the SP family are expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report the detection of novel yeast proteins (S. cerevisiae, p180; S. pombe, p200) that specifically bind Rb-regulated promoter elements in vitro dependent on nucleotides that are also required for binding and trans-activation by SP family members in vivo. Our results indicate that the S. cerevisiae retinoblastoma control element-binding activity 1) requires zinc for association with DNA; 2) does not bind to SCB, MCB, or E2F sites in vitro; 3) is cell cycle-regulated in a SWI6-independent fashion; and 4) maximally stimulates retinoblastoma control element-mediated transcription in early- to mid-S phase. Taken together, these data suggest that p180 may regulate the transcription of a subset of yeast genes whose expression is coincident with the onset and/or progression of DNA replication.
Highlights
Functional inactivation of the retinoblastoma (Rb)1 protein is associated with the genesis of a number of human tumors, including retinoblastoma, osteosarcoma, and breast, bladder, and small cell lung carcinomas
Proteins That Bind Retinoblastoma Control Elements (RCEs) Are Expressed in S. cerevisiae and S. pombe—To determine whether S. cerevisiae and/or S. pombe express RCEbinding proteins (RCE-BPs), a 31-base pair oligonucleotide corresponding to the human c-FOS RCE was radiolabeled and employed with yeast extracts in protein-DNA binding (“gelshift”) assays
Wild-type oligonucleotides corresponding to RCEs within the TGF-1 and c-MYC promoters eliminated the slowest migrating protein-DNA complex when employed as competitors in parallel protein-DNA binding assays
Summary
Retinoblastoma; RCP, retinoblastoma control protein; RCE, retinoblastoma control element; kb, kilobase pair(s); BUdR, bromodeoxyuridine. A 47-kDa factor in Saccharomyces cerevisiae that binds E2F sites in a cell cycle-dependent fashion has been reported, and a similar factor has been detected in Schizosaccharomyces pombe [36, 37] In concert with these findings, when expressed in S. cerevisiae Rb is phosphorylated by yeast cdks at sites that are targets of phosphorylation in mammalian cells [38]. Rb phosphorylation in yeast appears to be temporally controlled in a manner that is similar to that which occurs in mammalian cells; Rb is phosphorylated prior to the initiation of DNA synthesis in yeast coincident with the cell cycle checkpoint termed “Start” [27] Given these observations, it is tempting to speculate that yeast may harbor proteins functionally analogous to Rb that integrate progression of the cell cycle with transcriptional regulation. In this report we characterize the biochemical and functional properties of a novel cell cycle-regulated RCE-binding protein, p180, that is synthesized in S. cerevisiae and whose DNAbinding domain is functionally homologous to that of SP1, SP3, and perhaps other members of the SP family of mammalian transcription factors
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