Sequence requirements for combinatorial recognition of histone H3 by the MRG15 and Pf1 subunits of the Rpd3S/Sin3S corepressor complex

Abstract

The transcriptional output at a genomic locus in eukaryotes is determined, in part, by the pattern of histone modifications that are read and interpreted by key effector proteins. The histone deacetylase activity of the evolutionarily conserved Rpd3S/Sin3S complex is crucial for suppressing aberrant transcription from cryptic start sites within intragenic regions of actively transcribed genes. Precise targeting of the complex relies on the chromatin binding activities of the MRG15 (MRG stands for mortality factor on chromosome 4 related gene) and Pf1 subunits. Whereas the molecular target of the MRG15 chromodomain (CD) has been suggested to be H3K36me(2/3), the precise molecular target of the Pf1 plant homeodomain 1 (PHD1) has remained elusive. Here, we show that Pf1 PHD1 binds preferentially to the unmodified extreme N-terminus of histone H3 (H3K4me(0)) but not to H3K4me(2/3), which are enriched in the promoter and 5' regions of genes. Unlike previously characterized CD and PHD domains that bind to their targets with micromolar affinity, both MRG15 CD and Pf1 PHD1 bind to their targets with >100 μM affinity, offering an explanation for why both MRG15 CD and Pf1 PHD1 domains are required to target the Rpd3S/Sin3S complex to chromatin. Our results also suggest that bivalency, rather than cooperativity, is the operative mechanism by which Pf1 and MRG15 combine to engage H3 in a biologically significant manner. Finally, the studies reveal an unanticipated role of Pf1 PHD1 in engaging the MRG15 MRG domain, albeit in a Pf1 MRG-binding-domain-dependent manner, implying a key role for the MRG15 MRG-Pf1 MBD interaction in chromatin targeting of the Rpd3S/Sin3S complex.

Figure 1

MRG15 chromodomain is a methyllysine binding module. Titrations of ¹⁵N-labeled MRG15 CD with (a) H3K36me₃ (28–44), (b) H3K4me₂ (1-12), and (c) trimethyllysine. ¹H-¹⁵N correlated spectra of MRG15 CD were recorded as a function of increasing amounts of trimethyllysine or methyllysine-containing peptides in NMR buffer comprising 20 mM Tris-d₁₁ acetate-d₄ (pH 7.5), 1 mM DTT-d₁₀ and 0.2% NaN₃ at 25 °C. MRG15 CD concentrations used for the experiments in panels a-c were 0.2 mM except for panel b (0.75 mM). Chemical shift perturbations induced by H3K36me₃ (28–44) mapped on to the (d) polypeptide backbone and (e) molecular surface of the MRG15 chromodomain (PDB: 2F5K). The perturbations are color ramped from white (corresponding to 0 ppm) to yellow (average + 1 standard deviation = 0.055 ppm) to magenta (average + 3 standard deviations = 0.115 ppm). Side chains of residues that form the canonical methyllysine binding pocket are shown in panel d.

Figure 2

Pf1 PHD1 binds preferentially to unmodified histone H3. (a) SDS-PAGE analysis following a pulldown experiment conducted with GST-Pf1 PHD1 or GST-Pf1 PHD2 and an array of histone H3 peptides. The protein bands were visualized using Coomassie staining. ¹H-¹⁵N correlated spectra of ¹⁵N-labeled Pf1 PHD1 resulting from titrations with increasing amounts of (b) H3 (1-12) and (c) H3K4me₂ (1-12). All titrations were conducted at a concentration of 100 μM Pf1 PHD1 in NMR buffer comprising 20 mM Tris-d₁₁ (pH 7.0), 50 mM NaCl, 10 μM ZnSO₄, 15 mM DTT, and 0.2% NaN₃ at 25 °C. (d) Chemical shift perturbations induced by H3 (1-12) mapped on to the molecular surface of a homology model of Pf1 PHD1 based on the structure of the CHD4 PHD2 domain (PDB: 2L75). The perturbations are color ramped from white (corresponding to 0 ppm) to yellow (average + 1 standard deviation = 0.168 ppm) to magenta (average + 3 standard deviations = 0.338 ppm).

Figure 3

The extreme N-terminus of histone H3 engages Pf1 PHD1. (a) ¹H-¹⁵N correlated spectra of ¹⁵N-labeled H3 (1-42) recorded in the absence (black) and presence (red) of 2 equivalents of unlabeled Pf1 PHD1. The spectra were recorded at a concentration of 80 μM ¹⁵N-H3 in NMR buffer comprising 20 mM Tris-d₁₁ (pH 6.0), 50 mM NaCl, 10 μM ZnSO₄, 15 mM DTT, and 0.2% NaN₃ at 25 °C. Sequence-specific assignments are annotated. Minor peaks corresponding to resonances from minor conformers, presumed to result from cis-trans proline isomerization, are denoted by asterisks. (b) Backbone amide chemical shift deviations in the H3 peptide induced by Pf1 PHD1 graphed as a function of residue number. H3 resonances that disappeared upon PHD1 addition are denoted by stars while amide proton resonances in fast exchange with solvent are denoted by ‘x’s; prolines are denoted by ‘P’s.

Figure 4

Pf1 PHD1 binds histone H3 with similar affinities both in the absence and presence of MRG15 CD. Expanded plots of ¹H-¹⁵N correlated spectra showing perturbations induced by H3K36Cme₃ (1-42) of two Pf1 PHD1 correlations in (a) the absence and (b) the presence of an equimolar amount of ¹⁵N-MRG15 CD. Spectra recorded at molar ratios of H3K36Cme₃ (1-42) peptide:protein of 0:1, 1:1, 2:1, and 5:1 (green, light green, orange & red) are shown. Titrations were conducted in NMR buffer comprising 20 mM Tris (pH 7.5), 50 mM NaCl, 10 μM ZnSO₄, 15 mM DTT, and 0.2% NaN₃ at 25 °C. All spectra were recorded under identical solution conditions and were processed and displayed with the same parameters.

Figure 5

Pf1 PHD1 targets the MRG15 MRG domain. (a) ¹H-¹⁵N correlated spectra of ¹⁵N-PHD1/MBD in the absence (black) and presence (red) of an equimolar amount of full-length MRG15 (left panel) and MRG15 MRG (right panel). (b) ¹H-¹⁵N correlated spectra of ¹⁵N-PHD1 in the absence (black) and presence (green) of an equimolar amount of full-length MRG15 and in the presence of equimolar amounts of full-length MRG15 and Pf1 MBD (red). Pf1 PHD1 resonances that are relatively unperturbed upon MRG15 binding are identified. Titrations were conducted in NMR buffer comprising 20 mM Tris (pH 7.5), 50 mM NaCl, 10 μM ZnSO₄, 15 mM DTT, and 0.2% NaN₃ at 25 °C. All spectra were recorded under identical solution conditions and were processed and displayed with the same parameters.

Figure 6

A molecular model for bivalent recognition of histone H3 by the Pf1 and MRG15 subunits of the Rpd3S/Sin3S complex. The Pf1 PHD1 and MRG15 CD domains engage different segments of H3 in a non-cooperative manner but the two relatively weak interactions likely translate into a more efficient interaction due to the nanomolar affinity interaction between Pf1 MBD and MRG15 MRG that is bolstered by another interaction with the Pf1 PHD1 domain that collectively tether the two chromatin-binding domains in cis in the Rpd3S/Sin3S complex. Note that although the cartoon shows Pf1 and MRG15 contacting the same histone H3 polypeptide, it is conceivable that they contact different H3s in the same or adjacent nucleosomes.