Multivalent recognition of histone tails by the PHD fingers of CHD5

Abstract

The chromodomain, helicase, DNA-binding protein 5 (CHD5) is a chromatin remodeling enzyme which is implicated in tumor suppression. In this study, we demonstrate the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD(1) and PHD(2) alone and the tandem PHD(1-2) construct. Both domains displayed similar binding preferences for histone H3, where modification (e.g., methylation, acetylation, and phosphorylation) at H3R2, H3K4, H3T3, H3T6, and H3S10 disrupts high-affinity binding, and the three most N-terminal amino acids (ART) are crucial for binding. The tandem CHD5-PHD(1-2) displayed similar preferences to those displayed by each PHD finger alone. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5-PHD(1-2) simultaneously engages two H3 N-termini and results in a 4-11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin-binding modules function to interpret the combinatorial PTM capacity written in chromatin.

Figure 1. CHD3-5 protein architecture and sequence alignment

(A) A cartoon representation showing the N-terminal domain architecture of the highly similar CHD3-5. (B) A sequence alignment for the tandem PHD-fingers of CHD3-5. The first PHD-finger is highlighted in blue and the second in purple. Asterisks (*) denote invariant residues while colons (:) represent conserved residues.

Figure 2. The CHD5-PHD₁ and CHD-PHD₂ individual domains recognize H3unmod

(A) Each numbered box represents the space in which a single peptide resides (numbered key to the right). Within each numbered box a peptide is arrayed 16 times in a 4×4 spot grid to account for surface and spotting irregularities. (B) The GST-based assay used to determine the binding specificity of the PHD-fingers. B = biotin; SA = streptavidin; 647 = Alexa Fluor-647 (C,D) Raw images for the PHD₁ and PHD₂ screens (left panels). Values in the bar graphs were obtained by averaging the signal from 16 replicate spots. The values were normalized to the highest intensity spot in each individual library screen. Standard deviation is represented with error bars. (E,F) Discrimination factors for PHD₁ (E) and PHD₂ (F). Chi-squared values for each residue were calculated. Serine and threonine residues allow for 1 degree of freedom (DF), whereas lysine and arginine allow for 4 degrees of freedom (DF). Chi-squared values above the 99.9% confidence level for statistical significance are denoted with a single asterisk (*) along the x-axis (1 DF = 10.83 and 4 DF = 18.47).

Figure 3. The CHD5-PHD₁-₂ dual domain recognizes the unmodified histone H3 N- terminus

(A) Values for CHD5-PHD_1-2 were obtained by averaging the signal from the 16 replicate peptides arrayed on the slide surface. The values were normalized to the highest intensity spot in each individual library screen and standard deviation was calculated based on variation in signal intensity from the replicate 16 arrayed spots. (B) Discrimination factors for the modification preferences of CHD5-PHD_1-2 as previously described (46). (C) The methylation states at H3K4 and H3R2 were analyzed in the context of each other. Peptides containing only Rme2a/s were analyzed to determine their levels of methylation at H3K4 (frequency of methylation states in bottom circle graphs). Peptides containing only Kme2/3 were analyzed to determine the methylation levels at H3R2 (frequency of methylation states denoted in top circle graphs).

Figure 4. ALPHA assay with CHD5-PHD₁-₂ and H3unmod peptides

(A) Two identical peptides pertaining to the first eleven amino acids of the H3 N-terminus were functionalized with either biotin or 6×-histidine at their C-terminus to facilitate interaction with the ALPHA donor and acceptor beads respectively. Signal from the ALPHA depends on the CHD5-PHD_1-2 dependent proximity of a streptavidin-functionalized donor bead and Ni²⁺-chelated acceptor bead. (B) The condition for each ALPHA experiment is labeled along the x-axis. The normalized signal intensity is represented on the y-axis and is the average of three independent experiments. The standard deviation for the three experiments is represented with error bars.

Figure 5. NMR titration of CHD5-PHD₁-₂ and H3unmod

An overlay of the ¹H,¹⁵N HSQC spectra of PHD_1-2 as increasing amounts of the histone H3 tail peptide (1-12) are titrated in. Spectra are color coded according to the molar ratio of protein: peptide (see inset). Resonances and shifts (denoted by arrows) associated with PHD₁ are labeled in blue and those for PHD₂ are labeled in red. Overlays of the ¹H, ¹⁵N HSQC spectra of PHD_1-2, PHD₁, and PHD₂ in the unbound state (Supplementary Figure 4) and bound state (Supplementary Figure 5) are provided in Supplementary data.

Figure 6. A spatially addressed dilution library with H3unmod peptide

(A) A histone H3 peptide (1-12) was synthesized on modified cellulose discs. The peptide-cellulose conjugate was dissolved in DMSO, diluted over two orders of magnitude, and subsequently arrayed four times in duplicate on a microscope slide surface. (B) The binding ability of CHD5-PHD₂ compared with PHD₁ + PHD₂ and linked PHD_1-2 was interrogated with four replicate dilution libraries using a GST-based fluorescent assay. (C) The signal from the four replicate libraries was averaged and plotted against relative cellulose-peptide concentration. Squares represent 0.01 μM PHD_1-2 (■), open circles represent 0.01 μM PHD₂ (○), and triangles represent 0.01 μM PHD₁ + 0.01 μM PHD₂ (▲).