Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Abstract

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.

Keywords: PHD finger; chromatin; histone deacetylase (HDAC); histone modification; transcription.

FIGURE 1.

Subunit-interacting network analysis suggests two copies of the essential Rco1 subunit within Rpd3S. A–D, Coomassie staining of the indicated recombinant Rpd3S complexes that were produced in a baculovirus expression system. The bands corresponding to each subunit were indicated either as green dots (the tagged subunits for purification) or red dots (untagged subunits). Indicated Rpd3S complexes were prepared via FLAG purification. − indicates that the particular virus was omitted from the reconstitution. Asterisks indicate degradation or contaminated proteins. E, Eaf3 is in a monomeric form in Rpd3S. The indicated viruses were co-infected to insect cells. Western blots were performed after FLAG purification. F, Rpd3S contains two copies of Rco1. Recombinant Rpd3S was prepared through FLAG and HA tandem purification and stained with Coomassie Blue. G, a multiple sequence alignment of Rco1 orthologs. The following sequences were used: SpCph2 (Q09698, Schizosaccharomyces pombe), SpCph1(Q09819, S. pombe), SjCph2(SJAG_01110, Schizosaccharomyces japonicas), SjCph1(SJAG_03884, S. japonicas), SkuRco1 (protSku1303, Saccharomyces kudriavzevii), SbaRco1 (Sbay_66.15, Saccharomyces bayanus), ScRco1 (Q04779, Saccharomyces cerevisiae), and SpaRco1 (protSpa836, Saccharomyces paradoxus). Conserved residues are colored with the ClustalX scheme in Jalview (56). The resulting graph was further condensed and is displayed. Conservation value and phylogenetic tree were also calculated in Jalview. The domain structure of Rco1 is illustrated at the bottom, and the critical boundary residues were labeled, although the sequence gaps within Rco1 were not removed.

FIGURE 2.

Rco1 forms a homodimer within Rpd3S. A, co-immunoprecipitation showing that differently tagged Rco1 proteins associate with each other in yeast. Whole cell extracts (WCE) were prepared from yeast strains YBL766 (Rco1-TAP), YBL770 (Rco1-HA), and YBL772 (Rco1-TAP/Rco1-HA) and then immunoprecipitated (IP) using IgG-Sepharose before Western blotting. B, recombinant Rpd3S complex contains two copies of Rco1 that are independent of CHD and PHD1. FLAG and HA tandem purification was carried out for indicated Rpd3S complexes. CBB, Coomassie Brilliant Blue; WB, Western blot. C and D, Rco1 forms a homodimer in the absence of Eaf3 in a baculovirus expression system. E, an updated model of the Rpd3S-interaction network.

FIGURE 3.

Both copies of Rco1 are essential for Rpd3S nucleosome binding. A, purification of Rpd3S that contains two different copies of Rco1. IP, immunoprecipitation; CBB, Coomassie Brilliant Blue. B, working models of mutant Rpd3S complexes. C and D, EMSA showing that Rpd3S nucleosome binding was compromised when one SID domain was deleted. Um stands for unmodified nucleosomes, Me represents MLA-H3K36me3 nucleosomes. C, mono-nucleosomes. D, di-nucleosomes.

FIGURE 4.

Domain contributions to the full HDAC activity of Rpd3S. A, silver staining of native Rpd3S complexes purified from yeast. B, an experimental scheme of nucleosome-based HDAC assays. C and E, nucleosome-based HDAC assays using indicated Rpd3S complexes. The deacetylation activity, which was indicated by the amount of free ³H release, was plotted against the concentration of Rpd3S. Quantification was based on triplicates. Data are represented as the mean ± S.E. D and E, different Rpd3S complexes were tested in parallel with unmodified nucleosomes (D) and H3K36me3 nucleosomes (E).

FIGURE 5.

Structural insights from the DXMS analysis of Rpd3S in a free form. A, one-dimensional ribbon maps of deuterium-exchange profiles of the Rco1 subunits. AI indicates the autoinhibitory domain of Rco1. Colored bars represent the levels of deuterium incorporation at each residue, which range from 10% (blue) to 90% (red). Higher incorporation suggests faster H/D exchange and more solvent accessibility at the region. The secondary structure of Rco1 was predicted using PSIPRED v3.3. The conservation map was created using Jalview with all insertions in Rco1 being removed. The conservation value of each residue was also calculated in Jalview based on the sequence alignment of 24 Rco1 orthologs (all protein sequences were downloaded from PhylomeDB) (57): Phy004F6UM_1071379 (XBLA0G02280), Phy0002MXO_CANAL (Q5AH34), Phy004FE2O_KAZAF (H2AMM9), Phy000D24Z_SCHPO (Q09698), Phy000CZ77_YEAST (Q04779), Phy004FC3S_TETPH (G8BQR0), Phy000NOYU_SACBA (Sbay_556.6), Phy00244J2_ZYGRO (ZYRO0B02156g), Phy003M1NO_51660 (vKLBA.00009-j46), Phy000JL59_VANPO (Kpol_1002.11), Phy00241AL_LACTH (KLTH0D07040g), Phy0008MBP_KLULA (Q6CIK9), Phy000JQLI_KLUWA (Kwal_26.8059), Phy0023Y26_SACKL (SAKL0A09636g), Phy003GDAD_DEBHA (Q6BKT6), Phy0000COD_ASHGO (Q750N1), Phy004FZ60_HANAN (Wican1_61720), Phy000NTVT_SACCA (Scas_717.12), Phy003LTR2_51914 (vCACA.00021-j494), Phy00044F8_CANGA (CAGL0J01529g), Phy004F4JR_588726 (XNAG0C05760), Phy004FVLU_TORDC (TDEL0A03130), Phy004C1HG_PICST (A3LWC4), and Phy004FIKZ_NAUDC (G0WEU8). B, ribbon maps of the Eaf3 subunit after indicated incubation times. CHD represents the chromo barrel domain of Eaf3. The DBR (DNA binding region) indicates the DNA binding region of Eaf3. C, deuterium exchange results of CHD were mapped to the structure of free CHD (PDB code 3E9G) using PyMOL. Four aromatic residues that form the methyl-lysine binding pocket when CHD binds to K36me2 peptides are labeled. D, deuterium exchange results of MRG were mapped to a molecular model of MRG/SID based on PDB code 2LKM (4). SID was labeled in plain dark gray.

FIGURE 6.

Rco1 plays multiple roles in controlling Rpd3S functions. A, an illustration of Rco1 mutant constructs. FL, full-length. B and C, the N-terminal and C-terminal regions of Rco1 are important for suppressing cryptic transcription at the STE11 gene. B, all Rco1-expressing plasmids were transformed into the yeast strain YCR239 (the STE11:HIS3 reporter) (4). The yeast were serially diluted and spotted on the indicated plates. C, deletion of the N-terminal or C-terminal region of Rco1 does not compromise the binding of Eaf3 to Rpd3S. Whole cell extracts from the yeast strains in B were immunoprecipitated (IP) using anti-FLAG antibody and then subjected to Western blots. D and E, deletion of the N-terminal or C-terminal region of Rco1 did not disrupt complex integrity but abolished Rpd3S nucleosome binding. D, Coomassie staining of Rpd3S complexes purified from the baculovirus system. E, EMSA using mono-nucleosomes. F, Rco1 can still dimerize when the C-terminal region is deleted. The experimental scheme is indicated on the left. Two differently tagged Rco1 were used in a co-immunoprecipitation assay in a baculovirus system. WCE, whole cell extracts. G, the N-terminal and C-terminal truncated Rco1s can interact with full-length Rco1.