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

Abstract

Cbf1p is a basic-helix-loop-helix-zipper protein of Saccharomyces cerevisiae required for the function of centromeres and MET gene promoters, where it binds DNA via the consensus core motif CACRTG (R ‫؍‬ A or G). At MET genes Cbf1p appears to function in both activator recruitment and chromatin-remodeling. Cbf1p has been implicated in the regulation of other genes, and CACRTG motifs are common in potential gene regulatory DNA. A recent genome-wide location analysis showed that the majority of intergenic CACGTG palindromes are bound by Cbf1p. Here we tested whether all potential Cbf1p binding motifs in the yeast genome are likely to be bound by Cbf1p using chromatin immunoprecipitation. We also tested which of the motifs are actually functional by assaying for Cbf1p-dependent chromatin remodeling. We show that Cbf1p binding and activity is restricted to palindromic CACGTG motifs in promoter-proximal regions. Cbf1p does not function through CACGTG motifs that occur in promoter-distal locations within coding regions nor where CACATG motifs occur alone except at centromeres. Cbf1p can be made to function at promoter-distal CACGTG motifs by overexpression, suggesting that the concentration of Cbf1p is normally limiting for binding and is biased to gene regulatory DNA by interactions with other factors. We conclude that Cbf1p is required for normal nucleosome positioning wherever the CACGTG motif occurs in gene regulatory DNA. Cbf1p has been shown to interact with the chromatin-remodeling ATPase Isw1p. Here we show that recruitment of Isw1p by Cbf1p is likely to be general but that Isw1p is only partially required for Cbf1p-dependent chromatin structures.

Derepression ofINO1Transcription Requires Cooperation between the Ino2p-Ino4p Heterodimer and Cbf1p and Recruitment of the ISW2 Chromatin-Remodeling Complex

“…Our results show that Cbf1p and Ino2p-Ino4p bindings are interdependent. Cbf1p plays an important role in chromatin remodeling (25,52,70,71). Consistent with this role, we show that Cbf1p is required for the binding of the ISW2 chromatin-remodeling complex to INO1 upstream sequences bound by the Ino2p-Ino4p heterodimer.…”

“…bHLH transcription factors regulate transcription by binding DNA (42,77). As stated above, Ino2p and Ino4p interact with the INO1 promoter at two E boxes (UAS INO elements) (2,67,73), and Cbf1p activates the expression of MET genes by binding their promoters (52). Since Cbf1p regulates INO1 transcription, ChIP was used to determine if Cbf1p binds to the INO1 promoter.…”

“…Chromatin immunoprecipitation (ChIP) assays were performed as previously described (3), with some modifications. Cells were fixed with formaldehyde for 15 min for the INO2-TAP and INO4-TAP strains and 2 h for the CBF1-TAP, ino2⌬ CBF1-TAP, ino4⌬ CBF1-TAP, ISW2-TAP, and cbf1⌬ ISW2-TAP strains (52). Lysis was performed with a Multivortexer using glass beads.…”

Regulation of yeast oscillatory dynamics

“…2c, gray box) where the peak concentrations of MET4, MET28, MET30, and MET32 transcripts occurred in a narrow temporal window (Ϸ4 min). Cbf1p activates this complex by transcription and physical interaction to regulate sulfur assimilation (19,20). The transcription factor activity of Cbf1p, Met4p, Met28p, Met31p, and Met32p peaked Ϸ4-8 min after the maximum transcript concentration.…”

Multiple bHLH proteins regulate CIT2 expression in Saccharomyces cerevisiae

“…ENO1 ) (Nishi et al, 1995;Sato et al, 1999) and is also required for Ty1-mediated gene expression (Lohning and Ciriacy, 1994). Cbf1p binds to several MET gene promoters to regulate their transcription (Ferreiro et al, 2004;Kent et al, 2004;Kuras et al, 1997;Kuras and Thomas, 1995). Cbf1p also functions in chromosome segregation (Cai and Davis, 1990;Foreman and Davis, 1993) and DNA repair (Ferreiro et al, 2004).…”