Evidence has recently been mounting suggesting that a number of chromatin components previously thought to primarily or exclusively have structural function, also have a regulatory role in eukaryotic transcription. Notably, in yeast, histone H4 N-terminal sequence has been shown to be required for promoter activation of certain genes in vivo, and mutations in histone H3 (SIN2) or in SIN1 (which has some sequence similarity to HMG1) are able to suppress swi1, swi2, and swi3 mutations, restoring transcription to HO as well as a number of other genes. In this paper we report the identification of a novel protein or protein complex that specifically binds a short sequence in the HO regulatory region on the one hand, and on the other somehow appears to contact the SIN1 protein. We have shown that the DNA binding activity itself does not contain SIN1, since extracts from sin1 delta strains retain the activity. Interestingly, extracts made from cells carrying the dominant sin1-2 point mutation lack the binding activity. Furthermore, bacterially produced sin1-2 protein can dissociate a DNA/protein complex while a similarly produced SIN1 protein has no effect on the complex at similar concentrations. When the DNA sequence to which the protein complex binds is placed in a CYC1 promoter lacking a UAS (upstream activating sequence), it can serve as a weak UAS in a SIN1 dependent way. Our data imply that a sequence specific DNA binding protein(s) may mediate between the SIN1 protein and the basal transcription apparatus transcribing HO.
A protein or protein complex has previously been identified in Saccharomyces cerevisiae which both binds a short DNA sequence in URS1 of HO and interacts with SIN1. SIN1, which has some sequence similarity to mammalian HMG1, is an abundant chromatin protein in yeast and is thought to participate in the transcriptional repression of a specific family of genes. SIN1 binds DNA weakly, though it has no DNA binding specificity. Here we address the nature of the interaction between SIN1 and the specific DNA binding protein(s) to HO DNA. We show that the isolated C-terminal region of SIN1 can interact in vitro with the DNA binding protein, causing a supershift in a gel mobility shift assay. Interestingly, inclusion of the region in SIN1 which contains two acidic sequences, precludes the binding of recombinant protein to the DNA/protein complex.
DNA in vitro [8]. Interestingly, sinl mutants are also defective Abstract The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional in proper segregation of chromosome III but not chromosome repressor of a family of genes. In addition, sinl mutants are V during mitosis [2]. We have shown in parallel work to that defective in proper mitotic chromosome segregation. In an effort described in this paper, that the N-terminal of SIN1 is capable to understand the basis for these phenotypes, we employed the of interaction with CDC23 [9], a protein known to control yeast two-hybrid system to identify proteins that interact with chromosome segregation during mitosis [10][11][12]. SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein Here we extend our previous studies to help explain the belonging to the 'AAA' family of ATPases, is able to directly biochemical basis for the disparate phenotypes of SIN1. We interact with SIN1. Furthermore, we show, using recombinant used the yeast two-hybrid system [13][14][15] as a method to molecules in vitro, that a short 27 amino acid sequence near the identify proteins that directly associate with SIN1. We demon-N-terminal of SIN1 is sufficient to bind SAP1. Previous strate that a short 27 amino acid sequence near the N-termiexperiments defined different domains of SIN that interact with other proteins and with DNA. The C-terminal domain of SIN1 nus of SIN1 is able to directly interact with a novel protein we was shown to be responsible for interaction with a protein that have termed SAP1 (SIN1 _associating protein). The predicted binds the regulatory region of HO, a gene whose transcription is amino acid sequence of SAP1 indicates that it is a member of repressed by SIN1. The central 'HMGl-like region' of SIN1 the 'AAA' (ATPase _associated with diverse cellular _activities) binds DNA, while the N-terminal of SIN1 can bind CDC23, a family of proteins. protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 2. Materials and methods is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different Plasmids and yeast strains cellular functions.Plasmid pBTM 116/SIN1 was constructed by subcloning the EcoRI fragment from pGEX-3X/SIN1 [16] containing the SIN1 coding re-
The yeast SINI protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sinl mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SINI in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SINM. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SINI is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SINM to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SINM. Taken together with the results presented here, we suggest that SINM is a chromatin protein having at least a dual function: The N terminal of SINM interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SINM binds proteins involved in transcriptional regulation.
Recently it has become clear that general chromatin proteins as well as sequenco-specifle DNA b!nding protein, s are important in the control of gene expression. SIN1 in Saccharomyces cerevisiae is a chromatln component that regulates the transcription of a family of genes. Previously, we identified a 32 bp unique sequence (here termed XB$) in the promoter of one of those genes, HO, which s~eeifleally binds a protein that interacts with SINI. We also found that this sequence can function as a weak UAS in a beterologous promoter that is dependent on the presence of SINI. Here we report a relationship between the level of llO expreMIon and the presence of the short sequence in situ in the HO gene. By comparing the expression of llO from wild type or XB$ deleted HO promoters, we concluded that XBS serves as a weak UA$ in situ in the HO gene, that it influences HO transcription via the SWIlSNF complex, and that sequences other than the XBS mediate the affect of SINI on HO transcription. In addition, we show that a portion of the SINI protein that has sequence similarity to mammalian HMGI preferentially binds the XBS.
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