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. 2012 Jan 10;22(1):56-63.
doi: 10.1016/j.cub.2011.11.042. Epub 2011 Dec 15.

The Rpd3 Core Complex Is a Chromatin Stabilization Module

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Free PMC article

The Rpd3 Core Complex Is a Chromatin Stabilization Module

Xiao-Fen Chen et al. Curr Biol. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1. Rpd3S Inhibits RSC-dependent Nucleosome Eviction and Promotes Nucleosome Assembly In Vitro
(A) Silver stain gel of TAP-purified RSC2, Rpd3S and FACT complexes. (B) The effect of Rpd3S on RSC-dependent nucleosome remodeling. 2 nM RSC was incubated with 0.3nM 32P-labeled mononucleosome and 0, 13, 39 or 78 nM Rpd3S. The remodeling products were fractionated by native PAGE. A phosphorimage of the gel is shown. See also Figure S1A-C for the effect of H3K36me3 on Rpd3S in binding and RSC-mediated nucleosome remodeling reactions. (C) The effect of Rpd3S on RSC dependent nucleosome eviction. Left panel, 6 nM RSC was incubated with 0.3nM mononucleosome and 0, 13, 39 or 78 nM Rpd3S in the presence of 10 ng of pGEM3Z601R acceptor DNA. Bar graph on the right represents quantitation by ImageQuant TL (GE) of the 3 independent experiments. The relative amounts of free DNA generated by eviction were plotted as a bar graph normalized to that generated by 6 nM RSC alone, which was assigned a value of 100. The error bars show +/− standard deviation (SD). The P value is calculated using student’s t-test. See also Figure S1D for the effect of H3K36me3 on Rpd3S in RSC-mediated nucleosome eviction. (D) Rpd3S-mediated chromatin assembly assay. Left, the reaction contained 18 nM FACT, or 6, 12, 18 nM of Rpd3S respectively, with recombinant Xenopus octamers and a 32P-labeled 601 DNA fragment. A phosphorimage of a native gel is shown. Graph on the right represents quantitation of the amounts of assembled nucleosomes by Rpd3S relative to no Rpd3S control (i.e., octamers alone). The free DNA and assembled nucleosome are indicated. The error bars show +/− standard deviation (SD). The P value is calculated using student’s t-test. For chaperone assays see also Figure S1E for the effect of H3K36me3 on Rpd3S, Figure S1F for the effect of Rpd3S mutants and Figure S1G for the effect of trichostatin.
Figure 2
Figure 2. Rpd3L and 3-Subunit Core Complex Prevent RSC-dependent Nucleosome Eviction and Promote Nucleosome Assembly In Vitro
(A) Silver stain gel of TAP-purified Rpd3L. (B) The effect of Rpd3L on RSC-dependent nucleosome eviction. 6 nM RSC was incubated with 0, 15, 45, 90 nM Rpd3L, respectively, and analyzed as described in Figure 1C legend. (C) Nucleosome assembly with Xenopus octamers and 18 nM Rpd3L or 3-subunit core complex, respectively, as in Figure 1D legend. (D) Silver stain gel of recombinant 3-subunit core complex. The asterisk (*) indicates an unknown protein that co-purified with the 3-subunit core complex. (E) The effect of 3-subunit core complex on RSC-dependent nucleosome eviction. 6 nM RSC was incubated with 601 nucleosome and 0, 15, 45, 90 nM of recombinant core complex, respectively, and analyzed as in Figure 1C legend. See also Figure S2A for silver stained gels of the individual subunits, Figure S2B for their effect on RSC-mediated nucleosome eviction and Figure S2C for chaperone assays.
Figure 3
Figure 3. Rpd3 HDACs Inhibit RSC-Mediated Activation of Nucleosome Transcription
(A) Schematic of the C-tail template. The template contains the 601 positioning sequence and a 20-nucleotide single-stranded C-tail with an intervening polylinker from pGEM3Z601R. Pol II initiates from the C-tail. (B) The template was assembled into a mononucleosome with naïve recombinant Xenopus octamers and then preincubated with 3 nM RSC in the presence or absence of 30 or 60 nM Rpd3S, Rpd3L, 3-subunit core complex or FACT for 1h at 30°C. Pol II, α32P-CTP, NTPs and RNase H were then added for 15 min at 30°C. The 32P-labeled RNA products were fractionated on a 10% polyacrylamide/urea gel. A phosphorimage of the gel is shown. (C) Same assay as performed in (B), except that naked C-tail DNA template was used instead of nucleosomal template. (D) In vitro transcription was performed on naive or H3K36me3 nucleosomes. 3 nM RSC was incubated with 0, 5, 15, 45 nM Rpd3S or recombinant 3-subunit core complex, respectively.
Figure 4
Figure 4. Rpd3 Complexes Stabilize Chromatin In Vivo
(A) H3 levels were measured by ChIP in wild-type (WT), rpd3 H150A (H150A), and rpd3Δ (Vector) cells. ChIP DNA of Histone H3 and inputs were amplified, labeled, and hybridized to Agilent Tiling arrays. The average binding of 6,572 annotated genes and their upstream 500-bp regions are shown. Enrichment of H3 ChIP DNA is shown as the log2 ratios of ChIP versus input DNA. P values for the promoters were calculated using the Mann-Whitney test. See also Figure S3A for Rpd3 native vs. H150A protein levels, Figure S3B for global acetylation levels of H3K18 and H4K5 in Rpd3 native, H150A and null backgrounds, Figure S3C and S3D for Rpd3 association with Pol II and H3. (B) Box and whisker plot for two subsets of targets that have high or low Rsc2 enrichment. Each subset has 495 and 506 targets, respectively. The P values are calculated using student’s t-test. (C) H3 levels of the low Rsc2 targets were measured in wild-type, rpd3 H150A, and rpd3Δ cells. P values for the promoters were calculated using the Mann-Whitney test. (D) H3 levels of the high Rsc2 targets were measured in wild-type, rpd3 H150A, and rpd3Δ cells. P values for the promoters were obtained from the Mann-Whitney test.

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