Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor.

Abstract

A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay. This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000. An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000. CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S. cerevisiae and adjacent to the TATA element of the adenovirus major late promoter. A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.Centromeres are specialized regions of eucaryotic chromosomes that form sites of attachment for spindle microtubules in mitosis and meiosis (13,35,36). Little is known of the mechanism of spindle attachment or the molecules involved. We report here on the isolation of a centromere DNA-binding protein from Saccharomyces cerevisiae and its counterpart from human cells.Cloning and deletion analysis of yeast centromeres revealed three contiguous conserved sequences essential to or important for centromere function: centromere DNA element I (CDEI), an 8-base-pair (bp) conserved sequence; CDEII, an apparently random, adenine-plus-thymine (A + T)-rich stretch of approximately 90 bp; and CDEIII, a 25-bp conserved sequence (11,12,17,18,20,21,24,29,32,33,39). Nuclease digestion studies of chromatin containing centromere regions CEN3 and CENII revealed an unusual configuration: 250 bp, including the three conserved DNA elements, are especially protected from digestion (4). Crude fractions from S. cerevisiae bind naked DNA containing centromere sequences (5), but no resolution of the proteins involved has been described.A centromere-specific DNA-binding activity was discovered during recent studies of the GAL4-positive regulatory protein (7 MATERIALS AND METHODSPlasmid DNAs. Plasmid pG2p contains the 5' half of the GAL2 gene (7). Plasmids p181, p113, p200, p201, p203, p202, and p182 containing HindlIl fragments of CENI, CEN4, CEN7, CENII, CEN14, CEN15, and 2,um-STB in YRpJ41ARS1 were kindly provided by P. Hieter (20).Yeast strains and media. BY2 (6) is a protease-deficient strain (pep4-3) carrying the GAL4 gene on a high-copynumber plasmid. YNN 267 (a ura3-52 Ahis3-200 ade2-101 lys2-801 met-Agal4-537) was a gift from M. Johnston. BJ926 (a trpl prcl -126 pep4-3 prbl -1122 can1la his] prcl -126 pep4-3 prbl-1122 can]) was kindly provided by E. Jones. Cells were grown in YP medium (1% yeast extract, 2%Bacto-Peptone) containing 2% glucose to an A6w of 5.Nitrocellulose filter-binding assay and DNase I footprints. Plasmid pG2p DNA was linearized by cleavage with EcoRI, and the recessed 3' ends were fille...

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

“…We examined both palindromic CACGTG motifs and non-palindromic CACATG motifs. Motifs within the GAL2, GSH1, and SHU1 loci were chosen based on reports of Cbf1p-dependent changes in transcription (5,19,37,45). The remaining loci, DRS2, GDH3, PSK1, FUN30 and ERV46 (46 -50), were chosen at random from the left arm of chromosome I with the exception of GAL3 (51) on chromosome IV, which was chosen because it is associated with three CACATG motifs.…”

“…shown to bind to yeast centromere DNA (5)(6)(7)(8) via the CACRTG motif, which is found within the consensus centromere DNA Element 1 (CDEI) 1 RTCACRTG. Deletion of the CBF1 gene or deletion of CDEI motifs from centromeric DNA led to similar defects in mitotic and meiotic centromere function, indicating that Cbf1p is an important component of the centromerekinetochore complex (9,10).…”

Transcriptional control of the human biliary glycoprotein gene, a CEA gene family member down‐regulated in colorectal carcinomas

“…Deletion mutants placing the BGP Alu sequences or the two direct repeats in the upstream region immediately adjacent to the minimal promoter did not affect luciferase expression either, but the effects of these elements may be position dependent. [53]; AP-2 (conl), AP-2 consensus sequence 1 [53]; AP-2 (con2), AP-2 consensus sequence 2 [64] ; GAL2, the USF site in the GAL2 gene [63]; USF consensus sequence 1401; 01-AT, the HNF-4/LF-A1 site in the al-antitrypsin gene [52]; HNF-4 consensus sequence [41]; LF-A1 consensus sequence [52].…”

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

“…CDEII seems to be also necessary for optimal centromere activity but so far no protein has been reported that binds to CDEII. However, several proteins that bind to CDEI and CDEIII have been identified (Bram and Kornberg, 1987;Baker et al, 1989;Cai and Davis, 1990;Lechner and Carbon, 1991). These proteins are named centromere-binding factors (Cbf) and assemble into a centromere complex.…”

“…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).…”