CPF1, a yeast protein which functions in centromeres and promoters.

Abstract

Centromeres and several promoters of Saccharomyces cerevisiae contain a highly conserved octanucleotide, RTCACRTG, called CDEI. Using biochemical, genetic and structural analyses, we show that the same protein binds in vivo to CDEI sites in centromeres and in promoters. This protein, called CPF1 for centromere promoter factor, binds DNA as a dimer. Inactivation of the gene is not lethal but leads to a partial loss of the centromere function and to a Met‐ phenotype. Changes of the chromatin structure due to inactivation of CPF1 are seen at centromeres and at several CDEI‐carrying promoters (e.g. MET25, TRP1, GAL2). However promoter activities are affected in diverse ways making it presently difficult to describe a function for CPF1 in gene expression. The sequence of the cloned gene reveals in the carboxy‐terminal part two potential amphipathic helices preceded by a positively charged stretch of amino acids very similar to the helix‐loop‐helix domains recently identified in factors controlling tissue specific transcription in higher eukaryotes. Carboxy‐terminal truncations of CPF1 lacking this domain no longer bind to CDEI. The amino‐terminal half of CPF1 carries two clusters of negatively charged amino acid residues. Surprisingly, deletions of these clusters still render cells Met+ and lead only to a marginal decrease in centromere activity.

Cbf1p Is Required for Chromatin Remodeling at Promoter-proximal CACGTG Motifs in Yeast

“…Isw1p-Myc, which is fully functional in chromatin remodeling, is described in more detail in Kent et al (40). Disruptions of the CBF1 locus were created by gene replacement as described (19). For chromatin analysis in Figs.…”

“…Previous work has concentrated on potential Cbf1p binding motifs that occur in a gene regulatory context; a recent series of genome-wide protein localization experiments tested the binding of a variety of transcription factors to yeast promoter regions and suggested that 83% of intergenic CACGTG palindromes were likely to be bound by Cbf1p (31). Mutation of the CBF1 gene or mutation of potential CACRTG binding motifs in the regulatory DNA of the GAL2, TRP1, CYT1, PGK, RPL45, QCR8, and GSH1 genes leads to perturbation of transcriptional regulation (19,(32)(33)(34)(35)(36)(37). In addition, altered patterns of nuclease accessibility in chromatin consistent with changes in nucleosome position have been reported in association with Cbf1p binding at the TRP1 and QCR8 promoters (19,28,38), suggesting that chromatin remodeling is a general function of DNA-bound Cbf1p.…”

Characterization of promoter regions involved in high expression of KlCYC1

“…Although KlQCR8 and KlCYC1 are functionally related genes, regulation by KlCpf1p seems to operate over them in a different way since, in the KlCYC1 promoter, the KlCpf1-binding site is included into a negative regulatory region. Opposite effects of Cpf1p on transcriptional regulation of different genes are not unexpected; in S. cerevisiae, Cpf1p may act as an activator or as a repressor of transcription in different promoters [24]). Directed mutagenesis of each of the three Cpf1p binding consensus at the KlCYC1 promoter, or combinatorial mutation of them, as well as availability of viable KlCpf1p mutants will be necessary to obtain a reliable picture of the detailed influence of KlCpf1p on KlCYC1 expression.…”

The centromere‐binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae

“…Cbf1 is a helix-loop-helix leucine zipper protein that binds as a homodimer directly to the CDEI-DNA element of the centromere (Bram and Kornberg, 1987;Baker et al, 1989;Mellor et al, 1990). Functional studies in haploid S. cerevisiae cells showed that Cbf1p is non-essential (Cai and Davis, 1990).…”