Recruitment of Tup1-Ssn6 by yeast hypoxic genes and chromatin-independent exclusion of TATA binding protein

The Tup1-Ssn6 general repression complex in Saccharomyces cerevisiae represses a wide variety of regulons. Regulon-specific DNA binding proteins recruit the repression complex, and their synthesis, activity, or localization controls the conditions for repression. Rox1 is the hypoxic regulon-specific protein, and a second DNA binding protein, Mot3, augments repression at tightly controlled genes. We addressed the requirements for Tup1-Ssn6 recruitment to two hypoxic genes, ANB1 and HEM13, by using chromatin immunoprecipitation assays. Either Rox1 or Mot3 could recruit Ssn6, but Tup1 recruitment required Ssn6 and Rox1. We also monitored events during derepression. Rox1 and Mot3 dissociated from DNA quickly, accounting for the rapid accumulation of ANB1 and HEM13 RNAs, suggesting a simple explanation for induction. However, Tup1 remained associated with these genes, suggesting that the localization of Tup1-Ssn6 is not the sole determinant of repression. We could not reproduce the observation that deletion of the Tup1-Ssn6-interacting protein Cti6 was required for induction. Finally, Tup1 is capable of repression through a chromatin-dependent mechanism, the positioning of a nucleosome over the TATA box, or a chromatin-independent mechanism. We found that the rate of derepression was independent of the positioned nucleosome and that the TATA binding protein was excluded from ANB1 even in the absence of the positioned nucleosome. The mediator factor Srb7 has been shown to interact with Tup1 and to play a role in repression at several regulons, but we found that significant levels of repression remained in srb7 mutants even when the chromatin-dependent repression mechanism was eliminated. These findings suggest that the repression of different regulons or genes may invoke different mechanisms.     

Dynamics of gene targeting and chromatin remodelling by nuclear receptors

Activation of the murine-mammary-tumour virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). We have reconstituted this nucleoprotein transition with chromatin assembled on MMTV LTR DNA with Drosophila embryo extracts, purified GR, and HeLa nuclear extract. Chromatin remodelling in vitro is ATP-dependent and maps to a region identical with that found in vivo. We demonstrate specific, glucocorticoid response element dependent, binding of purified GR to a large, multi-nucleosome MMTV chromatin array and show that GR-dependent chromatin remodelling is a multistep process. In the absence of ATP, GR binds to multiple sites on the chromatin array and inhibits nuclease access to GR recognition sites. On the addition of ATP, GR induces remodelling resulting in a large increase in access of enzymes to their sites within the transition region. These findings are complemented by studies in living cells; using a tandem array of MMTV-Ras reporter elements and a form of GR labelled with the green fluorescent protein, we have observed direct targeting of the receptor to response elements in live mouse cells. Whereas the ligand-activated receptor is associated with the MMTV promoter for observable periods, photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. The results both in vitro and in vivo are consistent with a dynamic model ('hit and run') in which GR first binds to chromatin after ligand activation, recruits a remodelling activity and is then lost from the template.     

The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome

In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains.     

絮凝基因FLO1及FLO1c高表达提高工业酿酒酵母乙酸耐受性及发酵性能

乙酸是生物质乙醇发酵过程中酵母细胞面临的重要抑制剂之一,对细胞生长及发酵性能有强烈的抑制作用。增强酵母菌对乙酸胁迫的耐受性对提高乙醇产率具有重要意义。用分别带有完整絮凝基因FLO1及其重复序列单元C发生缺失的衍生基因FLO1c的重组表达质粒分别转化非絮凝型工业酿酒酵母CE6,获得絮凝型重组酵母菌株6-AF1和6-AF1c。同时以空载体p YCPGA1转化CE6的菌株CE6-V为对照菌株。与CE6-V相比,絮凝酵母明显提高了对乙酸胁迫的耐受性。在0.6%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.56倍和1.62倍;在1.0%(V/V)乙酸胁迫下,6-AF1和6-AF1c的乙醇产率分别为对照菌株CE6-V的1.21倍和1.78倍。可见絮凝能力改造能明显提高工业酿酒酵母的乙酸胁迫耐受性及发酵性能,而且FLO1内重复序列单元C缺失具有更加明显的效果。