Chromatin Remodelers Fine-Tune H3K36me-Directed Deacetylation of Neighbor Nucleosomes by Rpd3S  Chul-Hwan Lee, Jun Wu, Bing Li  Molecular Cell  Volume 52, Issue 2, Pages 255-263 (October 2013) DOI: 10.1016/j.molcel.2013.08.024 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 52, 255-263DOI: (10.1016/j.molcel.2013.08.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 The Binding of Rpd3S to Dinucleosomes Is Sensitive to the Length of Linker DNA in a Nonlinear Fashion (A) A schematic illustration of dinucleosome templates. All nucleosomes are identical except for the length of linker DNA, and their positions were confirmed in (B). (B) Nucleosome positioning of reconstituted dinucleosomal templates was confirmed by Bgl1 restriction digestion. As DNA probes were 32P-labeled at the 5′ ends, only the left-side (601-L) nucleosomes and residual undigested dinucleosomes can still be visualized upon digestion. Their migration patterns indicated the correct translational positions of 601-L nucleosomes and the dinucleosomes. (C) The binding of Rpd3S to dinucleosomes with different linker DNA was measured by EMSA assays. (D) Quantification of the results from (C). Data are represented as mean ± SEM. Molecular Cell 2013 52, 255-263DOI: (10.1016/j.molcel.2013.08.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Histone H3K36 Methylation Regulates Rpd3S HDAC Activity toward Neighboring Nucleosomes within Proper Linker Distance (A) Preparation of heterodinucleosome templates. Gel-purified mononucleosomes (designated as Right or 601-R) containing either unmodified (white-filled) or K36me3 (black-filled) histone H3 were labeled with 3H acetyl-CoA using Ada2-TAP (H3 acetylation) and NuA4 (H4 acetylation). 601-R nucleosomes were ligated at a Bgl1 site to 601-L nucleosomes that carry various lengths of linker DNA. The resulting dinucleosomes were gel purified and directly applied to HDAC assays in (C)–(E) or end labeled using 32P-γATP for EMSA assays in (F)–(H). (B) Confirmation of proper nucleosome positioning of the heterodinucleosomes. The restriction analyses were performed similarly to that described in Figure 1B, except that in this case both ends of the DNA probes were labeled. Therefore, both digested mononucleosomes can be seen. Lanes 19–26 contain the mononucleosomes before they were ligated to form dinucleosomes, and serve as marks for the correct migration pattern of digested dinucleosomes. (C–E) Histone deacetylase assays showing that the HDAC activity of Rpd3S toward neighboring nucleosomes depends on the length of linker DNA. Data are represented as mean ± SEM. (F–H) The binding affinity of Rpd3S to each heterodinucleosome was measured using gel mobility shift assays. Data are represented as mean ± SEM. Molecular Cell 2013 52, 255-263DOI: (10.1016/j.molcel.2013.08.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Deacetylation Preference of Rpd3S on Dinucleosome Substrates (A) The strategy to generate heterodinucleosome templates in which each nucleosome contains an equal amount of acetyl-groups. During the first round of the acetylation reaction, 3H-labeled acetyl-CoA or cold acetyl-CoA was used for either 601-L nucleosomes or 601-R nucleosomes. Subsequently, additional acetylation was carried out for all mononucleosomes using cold acetyl-coA to reach saturated acetylation levels on all nucleosomes. After ligation and gel purification, the resulting two types of dinucleosomes are identical in total acetylation status except for 3H-labeling, depending on the reaction. (B) The acetylation levels of each mononucleosome after two rounds of acetylation were monitored by western blotting using anti-acetylated H3 or acetylated H4 antibodies. An antibody against histone H4 was used to demonstrate the loading consistency. (C) The final heterodinucleosomes prepared above were resolved on a 3.5% acrylamide gel, and the dinucleosome concentrations were quantified based on the amount of DNA. (D) Quantification of HDAC results based on three independent experiments. Data are represented as mean ± SEM. (E) A model for different mechanisms of action of Rpd3S on dinucleosomes containing different linker DNA. The thicknesses of the arrows reflect the strength of HDAC activity. Thirty base pairs (mid) is near optimal distance in that Rpd3S can efficiently deacetylate both methylated nucleosome and neighboring unmethylated nucleosome; 15 bp (left) represents the situation where two nucleosomes are too close and Rpd3S can function on neighboring nucleosomes but with less efficiency; at 70 bp (right) distance, Rpd3S can no longer deacetylate neighboring nucleosomes. Molecular Cell 2013 52, 255-263DOI: (10.1016/j.molcel.2013.08.024) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Chromatin Remodeling Factors Fine-Tune the Rpd3S HDAC Activity on Dinucleosomes by Altering Nucleosomal Spacing (A) The experimental design. (B) Rpd3S HDAC activity on dinucleosomes is enhanced by indicated chromatin remodeling complexes in an ATP-dependent manner. Data are represented as mean ± SEM. (C) Chromatin remodeling per se does not increase Rpd3S activity. Similar experiments were performed as in (B) except that mononucleosomes with indicated modification pattern were used as substrates. Data are represented as mean ± SEM. (D–F) ISWI family of ATPases mediates nucleosome sliding on dinucleosome templates. (D) Chromatin remodeling assay using dinucleosome substrates containing a 70 bp linker DNA. The reactions were directly loaded on a 3.5% native PAGE gel to resolve dinucleosome populations that are positioned differently. (E) Predicted positioning changes of each nucleosome caused by Isw1-TAP and ACF-mediated dinucleosome sliding. Restriction sites Not1 and Bgl1, which are just outside each nucleosome, are used to monitor nucleosome movement. (F) Restriction site protection assays for dinucleosome sliding suggest that both nucleosomes moved toward the center. Following the remodeling assay described above, the reaction mixtures were subjected to restriction digestion with NotI or BglI. The resulting digested and undigested nucleosomes were resolved on a 4% PAGE gel. Molecular Cell 2013 52, 255-263DOI: (10.1016/j.molcel.2013.08.024) Copyright © 2013 Elsevier Inc. Terms and Conditions