The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region

Abstract

Regulation of CLB2 is important both for completion of the normal vegetative cell cycle in Saccharomyces cerevisiae and for departure from the vegetative cell cycle upon nitrogen deprivation. Cell cycle-regulated transcription of CLB2 in the G2/M phase is known to be brought about by a set of proteins including Mcm1p, Fkh2/1p and Ndd1p that associate with a 35 bp G2/M-specific sequence common to a set of co-regulated genes. CLB2 transcription is regulated by additional signals, including by nitrogen levels, by positive feedback from the Clb2-Cdc28 kinase, and by osmotic stress, but the corresponding regulatory sequences and proteins have not been identified. We have found that the essential Reb1 transcription factor binds with high affinity to a sequence upstream of CLB2, within a region implicated previously by others in regulated expression, but upstream of the known G2/M-specific site. CLB2 sequence from the region around the Reb1p site blocks activation by the Gal4 protein when positioned downstream of the Gal4-binding site. Since a mutation in the Reb1p site abrogates this effect, we suggest that Reb1p is likely to occupy this site in vivo.

Figure 1

Diagram of the regulatory sequences of the CLB2 gene. Sequences from –863 to –627, called UAS, are sufficient to confer full expression on a reporter gene (18). The G₂/M-specific site from –698 to –643 is bound by Mcm1p, Fkh2p and Ndd1p and is found in a set of G₂/M-regulated genes. The Reb1p site at –784 was identified in the course of these experiments; the sequence protected from DNase I digestion (–795 to –770) as well as the Reb1p-binding site (underlined) and putative Mcm1p-binding site (overlined) are shown. DNA fragments used in the binding experiments in this study are illustrated here: CLB2-L1 (–933 to –514), CLB2-L2 (–933 to –622), CLB2-S (–698 to –662) and CLB2-R (–797 to –756).

Figure 2

DNA binding of the heparin–agarose-purified yeast proteins assessed by EMSA. (A) The binding activity of heparin–agarose-purified yeast proteins for CLB2-L1 DNA (–933 to –514), which contains the UAS sequences, and CLB2-S DNA (–698 to – 662) containing the Mcm1p/Fkh2p/Ndd1p-binding sites, as well as STE2 and CLN3 UAS sequences, containing known Mcm1p-binding sites. The CLB2 DNAs are illustrated in Figure 1. Bound and free DNAs are indicated by arrows; the free CLB2-S DNA is not shown. (B) Binding of yeast heparin–agarose-purified proteins to 5 × 10^–11 M labeled CLB2-L2 DNA (–933 to –622) was competed with unlabeled CLB2 DNA sequences from –825 to –622. Binding affinity was assessed by Scatchard analysis.

Figure 3

Identification of DNA sequences important for binding. (A) The binding site of the heparin–agarose-purified protein fraction was mapped by DNase I protection on each strand of the CLB2-L2 DNA fragment. The protected region and sequence on each strand are illustrated, as well as the position of the known G₂/M-specific sequences. (B) Binding and competition of binding with unlabeled oligonucleotides bearing the protected DNA sequence. DNA-binding activity was examined using EMSA with labeled CLB2-R containing 42 bp of CLB2 (–797 to –756). Oligonucleotides with either the wild-type sequence or mutations in the putative Mcm1p- and Fkhp-binding sites (shown in D) were also used as competitors. In each set, unlabeled competitors were at the following concentrations: 0, 2.5 × 10^–10, 5 × 10^–10, 7.5 × 10^–10 and 10 × 10^–10 M. (C) DNA binding to full-length CLB2-L2 (–933 to –622), assessed by EMSA, is competed by an oligonucleotide bearing only 26 bp of CLB2 (corresponding to the protected sequence –795 to –770). This oligonucleotide with a single mutation in the Reb1p (Reb1 site-m1) site fails to compete DNA binding. Competitor DNAs were present at 0, 0, 0.5 × 10^–10, 1 × 10^–10, 2.5 × 10^–10, 5 × 10^–10, 25 × 10^–10 and 50 × 10^–10 M. (D) DNA sequences of the protected region, the potential binding sites for Mcm1p, Fkh2p and Reb1p, and the mutations introduced into the oligonucleotides used in (B) and (C). For each putative binding site, the sequences that constitute a binding site are shown, with the most conserved bases in bold; the mutations introduced to impair binding are underlined.

Figure 4

Reb1 protein, translated in vitro, binds CLB2 upstream sequences. Reb1 protein (±[³⁵S]methionine) was made in a coupled in vitro transcription–translation system from a plasmid bearing the REB1 gene under control of the T7 promoter. Lanes 1–3: labeled CLB2-L2 DNA in EMSA using yeast heparin–agarose-purified proteins (lane 1), in vitro translated Reb1 protein (lane 2) and in vitro translated luciferase (lane 3). Lanes 4 and 5: ³⁵S-labeled Reb1 protein in EMSA using unlabeled CLB2-L2 DNA (lane 4) or no added CLB2 DNA (lane 5).

Figure 5

Alteration of either the amount or size of Reb1 protein produced in yeast alters the EMSA complex. (A) EMSA with labeled CLB2-R DNA and crude extracts made from a wild-type yeast strain (J47A) and a conditional reb1 mutant (J342) (deleted for the chromosomal reb1 gene and expressing REB1 under control of the Gal promoter) (36). Yeast were grown in galactose + glucose, then shifted to glucose for 4–4.5 generations to repress REB1. Growth in galactose alone is toxic to strain J342, presumably because overproduction of Reb1 protein is toxic (36). Lanes 1 and 2, extracts from the wild-type parent J47A grown in glucose (lane 1) or glucose + galactose (lane 2); lanes 3 and 4, extracts from J342, carrying the conditional P_GAL REB1 strain after the switch to glucose (lane 3) or grown in glucose + galactose (lane 4); lane 5, extracts from the wild-type EG573 yeast strain. (B) EMSA of complexes formed with labeled CLB2-R DNA using extracts derived from the J343 yeast strain expressing the 573 amino acid Reb1 protein homolog from K.lactis (lane 1) or from the J47A yeast strain expressing the 802 amino acid S.cerevisiae Reb1 protein (lane 2) (36).

Figure 6

The CLB2 Reb1 site modulates Gal4p activation of gene expression. (A) Diagram of the four reporter constructs that were integrated at the URA3 locus and assayed in (B). The Gal4p DNA-binding site and the a-specific genes (asg) operator are present in the original construct (37). The asg operator was replaced by CLB2 sequences from –942 to –721 (either wild type or bearing the Reb1p site m1 mutation) or by a linker sequence as shown. (B) The expression of the CYC1-lacZ gene from yeast strains bearing each plasmid was examined on plates containing X-gal and galactose.