The Rpd3 Core Complex Is a Chromatin Stabilization Module

Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1737, USA.
Current biology: CB (Impact Factor: 9.57). 12/2011; 22(1):56-63. DOI: 10.1016/j.cub.2011.11.042
Source: PubMed


The S. cerevisiae Rpd3 large (Rpd3L) and small (Rpd3S) histone deacetylase (HDAC) complexes are prototypes for understanding transcriptional repression in eukaryotes [1]. The current view is that they function by deacetylating chromatin, thereby limiting accessibility of transcriptional factors to the underlying DNA. However, an Rpd3 catalytic mutant retains substantial repression capability when targeted to a promoter as a LexA fusion protein [2]. We investigated the HDAC-independent properties of the Rpd3 complexes biochemically and discovered a chaperone function, which promotes histone deposition onto DNA, and a novel activity, which prevents nucleosome eviction but not remodeling mediated by the ATP-dependent RSC complex. These HDAC-independent activities inhibit Pol II transcription on a nucleosomal template. The functions of the endogenous Rpd3 complexes can be recapitulated with recombinant Rpd3 core complex comprising Sin3, Rpd3, and Ume1. To test the hypothesis that Rpd3 contributes to chromatin stabilization in vivo, we measured histone H3 density genomewide and found that it was reduced at promoters in an Rpd3 deletion mutant but partially restored in a catalytic mutant. Importantly, the effects on H3 density are most apparent on RSC-enriched genes [3]. Our data suggest that the Rpd3 core complex could contribute to repression via a novel nucleosome stabilization function.

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    • "and S3E–S3G). Rpd3 represses transcription through histone deacetylation (Kadosh and Struhl, 1998), can stabilize promoter nucleosomes (Chen et al., 2012), and has been implicated in stress response (Alejandro-Osorio et al., 2009; Weiner et al., 2012) and repression of ribosome biogenesis genes upon inactivation of TOR signaling (Huber et al., 2011; Humphrey et al., 2004; Rohde and Cardenas, 2003). In addition, Rpd3 is predicted to play roles in quiescence through genetic and computational analyses (Reimand et al., 2012). "
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    ABSTRACT: Quiescence is a conserved cell-cycle state characterized by cell-cycle arrest, increased stress resistance, enhanced longevity, and decreased transcriptional, translational, and metabolic output. Although quiescence plays essential roles in cell survival and normal differentiation, the molecular mechanisms leading to this state are not well understood. Here, we determined changes in the transcriptome and chromatin structure of S. cerevisiae upon quiescence entry. Our analyses revealed transcriptional shutoff that is far more robust than previously believed and an unprecedented global chromatin transition, which are tightly correlated. These changes require Rpd3 lysine deacetylase targeting to at least half of gene promoters via quiescence-specific transcription factors including Xbp1 and Stb3. Deletion of RPD3 prevents cells from establishing transcriptional quiescence, leading to defects in quiescence entry and shortening of chronological lifespan. Our results define a molecular mechanism for global reprogramming of transcriptome and chromatin structure for quiescence driven by a highly conserved chromatin regulator. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 08/2015; 59(5). DOI:10.1016/j.molcel.2015.07.014 · 14.02 Impact Factor
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    • "The lysates were collected by puncturing the bottom of a tube as described above followed by high speed centrifugation after treatment with DNase I and Heparin each for 5 min at room temperature. The protein was first purified by using IgG sepharose beads and TEV protease as previously described with minor modifications [29] and further purified with Ni-NTA Agarose (QIAGEN) using binding buffer (20 mM NaH2PO4, pH 7.4, 0.5 M NaCl, 10 mM Imidazole) and elution buffer containing 500 mM Imidazole. Finally, protein was dialyzed and concentrated in reaction buffer including 10% glycerol. "
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    ABSTRACT: Cathepsin L, a lysosomal protein in mouse embryonic stem cells has been shown to clip the histone H3 N- terminus, an activity associated with gene activity during mouse cell development. Glutamate dehydrogenase (GDH) was also identified as histone H3 specific protease in chicken liver, which has been connected to gene expression during aging. In baker's yeast, Saccharomyces cerevisiae, clipping the histone H3 N-terminus has been associated with gene activation in stationary phase but the protease responsible for the yeast histone H3 endopeptidase activity had not been identified. In searching for a yeast histone H3 endopeptidase, we found that yeast vacuolar protein Prb1 is present in the cellular fraction enriched for the H3 N-terminus endopeptidase activity and this endopeptidase activity is lost in the PRB1 deletion mutant (prb1Δ). In addition, like Cathepsin L and GDH, purified Prb1 from yeast cleaves H3 between Lys23 and Ala24 in the N-terminus in vitro as shown by Edman degradation. In conclusion, our data argue that PRB1 is required for clipping of the histone H3 N-terminal tail in Saccharomyces cerevisiae.
    PLoS ONE 02/2014; 9(2):e90496. DOI:10.1371/journal.pone.0090496 · 3.23 Impact Factor
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    • "Similarly, using a lexA-Ume6 reporter it was reported that Rpd3 may repress transcription independent of its histone deacetylase activity [48]. Moreover, the mechanism of repression apparently involves nucleosome stabilization by the Rpd3 core complex [49]. We postulated that diploid cells expressing catalytically inactive Rpd3 would exhibit a different phenotype than cells with a deleted RPD3 allele and phenotype similar to that of sds3Δ rco1Δ diploids. "
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    ABSTRACT: In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.
    PLoS ONE 12/2013; 8(12):e85088. DOI:10.1371/journal.pone.0085088 · 3.23 Impact Factor
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