Characterization of the ECB Binding Complex Responsible for the M/G₁-Specific Transcription of CLN3 and SWI4

Abstract

The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G 1 phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.Mcm1, its mammalian counterpart (serum response factor), and other members of the MADS box family of transcription factors share a 56-amino-acid MADS box, carrying a conserved DNA binding and dimerization domain (42). They are combinatorial transcription factors in that they typically bind adjacent to and derive their regulatory specificity from other DNA binding and/or accessory factors (3,20,42). Figure 1 depicts four classes of transcription complexes that include Mcm1. Depending upon the context, Mcm1 can be a component of an activating or a repressing complex, and these complexes can be regulated by specific events in the cell cycle or in response to internal or external cues. For example, repression of a-specific genes in ␣ haploid and a/␣ diploid cells involves cooperative binding of ␣2 and Mcm1 to adjacent binding sites (22,39). Then ␣2 recruits the Ssn6/Tup1 repressor complex to the site and prevents transcription (16). In contrast, ␣-specific genes are induced by Mcm1 and another ␣-specific transcription factor, ␣1 (43). In a cells, ␣2 is not produced, so the repressing complex cannot be formed. In that case, Mcm1 cooperates with another haploid-specific transcription factor, Ste12, to activate transcription and confer pheromone responsiveness to these promoters (12,14).Mcm1 is also involved in the regulation of arginine metabolism (not shown), where it forms a complex with Arg82 and two other DNA binding proteins: Arg80 and -81 (13, 33). The presence of arginine is sensed by Arg82 and results in the activation of transcription of catabolic enzymes and repression of anabolic enzymes, depending upon the context. Arg82 has recently been identified as an inositol-1,4,5-triphosphate kinase, and this discovery suggests a direct connection between the lipid signaling cascade and gene regulation in yeast (35).Mcm1 also plays a rol...

Transcriptional Analysis of the Candida albicans Cell Cycle

“…Nonetheless, the motifs in S. cerevisiae (CCAGCA) and in S. pombe (CCAGCC), bound in both cases by Ace2, represent highly species-specific signatures not seen in the phylogenetically intermediate C. albicans that relies on the consensus core five-nucleotide motif (CCAGC) for its Ace2p specificity. In addition to the Ace2 regulatory circuit, S. cerevisiae has a second M/G1 regulatory circuit involving the ECB (Mai et al, 2002) and the Mcm1p/Yox1p transcriptional regulatory complex (Pramila et al, 2002). Neither S. pombe nor C. albicans seem to possess this additional circuit, although both have Yox1 homologue and C. albicans has a Mcm1 orthologue (Tuch et al, 2008a).…”

Acetyl-CoA induces transcription of the key G1 cyclin CLN3 to promote entry into the cell division cycle in Saccharomyces cerevisiae

“…Mcm1p is another transcription factor reported to mediate CLN3 transcription in a cell-cycle-dependent manner (56)(57)(58)(59). We tested the binding of Mcm1p to the CLN3 promoter across the YMC.…”

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

“…RNA measurements: RNA levels were quantitated with S1 protection assays performed using oligonucleotide probes as described (Mai et al 2002). The probe sequences are available upon request.…”