Chromatin structure of altered yeast centromeres.

Abstract

We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomyces cerevisiae by using an indirect endlabeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-250 base pairs long, centered around the conserved centromere DNA element (CDE) III. A point mutation in CDE III that changes a central cytidine to a thymidine and completely disrupts centromere function has lost the chromatin conformation typically associated with the wild-type centromere. A second conserved DNA element, CDE I, is spatially separated from CDE HI by 78.86 A+T-rich base pairs, which is termed CDE H. The sequence and spatial requirements for CDE II are less stringent; alterations in CDE II length and sequence can be tolerated to a limited extent. Nuclease-resistant cores are altered in dimension in two CDE II CEN3 mutations. Two CDE I deletion mutations that retain partial centromere function also show nuclease-resistant regions of reduced size and intensity. The results from a number of such altered centromeres indicate a correlation between the presence of a protected core and centromere function.The centromere is a specific domain on eukaryotic chromosomes that plays an essential role in the faithful segregation of the chromosomes during mitotic and meiotic cell divisions. Cytologically, the centromere has been defined as the region of the chromosome that interacts with the spindle fibers during cell division. It has been proposed that centromerespecific DNA-binding proteins mediate the interaction between centromeres and spindle fibers. Following development of a functional assay for centromeres of the yeast Saccharomyces cerevisiae, the centromeres of 11 of the 16 chromosomes have been isolated (1-6). These centromere (CEN) sequences confer stable inheritance on autonomously replicating plasmids during mitosis and meiosis (1). Analysis and comparison ofthe DNA sequences of the 11 centromeres isolated has allowed description of several conserved sequence elements and their spatial organization (3,6,7). Each centromere has a highly conserved 8-base-pair (bp) In this report we have investigated the chromatin structure of several centromere mutations. We demonstrate that the nuclease-resistant chromatin core associated with the wildtype centromere is correlated with function. Specifically, point mutations in CDE III that destroy centromere function also destroy the nuclease-resistant region. The use of restriction enzymes as probes for chromatin structure confirms the change in structure associated with loss of centromere function. Centromere mutations that alter the distance between CDE I and CDE III alter the overall dimensions of the nuclease-resistant core. CEN mutations deleted for CDE I retain a residual nuclease-resistant structure that is reduced in intensity proportional to their loss of function. MATERIALS AND METHODSStrains. S. cerevisiae strain J17 (MATa, his2, adel, trpl, metl4...

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

“…At the yeast centromere, the position of the CDEI motif at one end of the core CEN sequence means that Cbf1p binds in close proximity to the centromere-specific nucleosome-like particle (10,15). Loss of Cbf1p causes a decrease in centromere efficiency during mitosis and leads to a subtle alteration in the nuclease accessibility surrounding the core particle (59,19). This is consistent with a role for Cbf1p in stabilizing the centromere core.…”

Chromatin Proteins Are Determinants of Centromere Function

“…A subset of these proteins bind specifically to the CDE elements present in centromeric DNA. For example, CDEI recruits the Cbf1 protein to mark the left boundary of the protected core domain (Saunders et al 1988;Bram and Kornberg 1987;Baker et al 1989;Cai and Davis 1989;Jiang and Philippsen 1989). Chromatin digestion of cbf1Δ cells results in a slightly reduced area of nuclease protection of the core centromere, and both CDEIΔ and cbf1 Δ cells display only minor defects in chromosome segregation (Cai and Davis 1990).…”

Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.

“…Single point mutations in CDEIII can abolish measurable centromere function and disrupt centromere chromatin structure (McGrew et al, 1986;Ng and Carbon, 1987;Hegemann et al, 1988;Saunders et al, 1988). A multisubunit complex (CBF3) that specifically binds to wild-type CDEIII DNA in vitro has been identified (Lechner and Carbon, 1991).…”

“…The relative contribution of each of these elements to mitotic fidelity has been assessed by extensive mutational analysis (reviewed in Hegemann and Fleig, 1993). In addition, in vivo, a unique nuclease-resistant chromatin structure (encompassing -200 bp) is associated with the centromere DNA -throughout the cell cycle (Bloom and Carbon, 1982;Saunders et al, 1988;Funk et al, 1989;Schulman and Bloom, 1991). In recent years, a combination of genetic and biochemical approaches has yielded a satisfying congruence in terms of identifying putative protein constituents of this chromatin structure, which presumably corresponds to the yeast centromere-kinetochore complex.…”

“…These results suggest that CDEII is somewhat flexible and might comprise iterations of a protein binding site. To date, no CDEII-specific DNA binding proteins have been identified, although both nuclease protection experiments (Bloom and Carbon, 1982;Saunders et al, 1988;Funk et al, 1989) and in vivo DMS footprinting experiments (Densmore et al, 1991) suggest that protein(s) are bound to CDEII in vivo. However, it remains possible that the intrinsic structural properties of homopolymeric A + T-rich DNA (e.g.…”