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 function in 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.It is now widely accepted that histone post-translational modifications (PTMs) 2 and other structural features of chromatin, such as variant histones and DNA modifications, mainly serve as signal platforms to direct downstream regulatory events (1, 2). In general, four major components of chromatin structure contribute to the signals that can be interpreted by downstream chromatin regulators. (i) Histone peptides that carry certain PTMs contribute. Specifically modified histone peptides can be recognized by protein domains/motifs that are commonly referred as PTM "readers." A large repertoire of such domain/PTM interactions have been documented, and they are essential for targeting downstream factors to a given genomic locus (3). Reader domains not only distinguish different types of modifications and the locations of PTMs but also the specific states of modifications. However, due to relatively weak affinity, it is unclear whether these interactions are sufficient to mediate the recruitment of chromatin complexes. For example, although histone H3 methylation at residue Lys-36 (H3K36me) is an essential signal for the histone deacetylase Rpd3S, the contact between the chromodomain of the Eaf3 subunit (CHD Eaf3 ) of Rpd3S and H3K366me only contributes to the binding specificity but not to the overall affinity (4). (ii) Histone globular domains contribute. Because PTMs are mostly cl...

Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity

“…Rpd3 Complex Isolation-Recombinant Rpd3S complexes were purified from a Sf21 insect cell-based baculovirus expression system as described previously (9,11). Briefly, freshly passed Sf21 cells were co-infected with individual virus that encodes each subunit of Rpd3S for 48 h. The cells were collected and lysed in BV lysis buffer (50 mM HEPES, pH 7.9, 300 mM NaCl, 2 mM MgCl 2 , 0.2% Triton X-100, 10% glycerol, 0.5 mM EDTA, and freshly added protease inhibitors) on ice for 30 min.…”

“…Rpd3S, an histone deacetylase that functions in a co-transcriptional manner, has five conserved chromatin-binding domains: a chromodomain in Eaf3, which recognizes Set2-mediated histone H3 lysine 36 methylation (H3K36me) (6 -8), and four plant homeodomains (PHDs), two per copy of Rco1, which has recently been shown to form a homodimer in Rpd3S (see Fig. 1A) (9). The chromodomain of Eaf3 and the N-terminal PHD finger of Rco1 (PHD1) have previously been characterized and are necessary for Rpd3S function and nucleosome engagement (6 -8, 10).…”

Shaping the cellular landscape with Set2/SETD2 methylation

“…Eaf3 contains a chromo domain that recognizes H3K36me2/3 [90–94], and Rco1 contains a dual plant homeodomain (PHD) finger motif that is necessary for chromatin engagement [95] and a homo-dimerization domain [96]. Both PHD fingers are essential for chromatin binding and Rpd3S function, and both domains prefer to bind the unmodified N-terminus of H3 [97].…”

“…Both PHD fingers are essential for chromatin binding and Rpd3S function, and both domains prefer to bind the unmodified N-terminus of H3 [97]. Interestingly, Rco1 exists as a dimer within the Rpd3S complex, adding two additional chromatin contacts to the complex [96]. Like the Isw1b complex, Eaf3’s recognition of H3K36me helps to localize the Rpd3S complex to the 3’-ends of gene bodies [90, 94, 95] where it can deacetylate histone tails.…”

Modulation of gene expression dynamics by co-transcriptional histone methylations

“…Eaf3 chromodomain directly binds to methylated histones on H3K36 and Rco1 PHD finger interacts with unmodified histone tails. 39 , 68 , 69 , 70 Rpd3S seems to be also recruited to transcribed regions by elongating RNA polII via interaction with the phosphorylated CTD, but histone deacetylation requires a direct interaction of this complex with methylated histones on H3K36. 66 , 67 Deacetylation by Rpd3S within coding regions slows RNA polII elongation ( Figure 4a ).…”