Combinatorial profiling of chromatin binding modules reveals multisite discrimination - PubMed (original) (raw)

Combinatorial profiling of chromatin binding modules reveals multisite discrimination

Adam L Garske et al. Nat Chem Biol. 2010 Apr.

Abstract

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.

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Conflict of interest statement

Competing financial interests

J.M.D and A.L.G have a patent pending (No. 11/585,625) that describes the construction and uses of PTM peptide libraries.

Figures

Figure 1

Figure 1. PTMs included in the 5000- member combinatorial H3 tail peptide library

(a) Positions of randomization are annotated by ‘X’. Arginines 2 and 8 can be unmodified, monomethylated, symmetrically or asymmetrically dimethylated or citrullinated. Lysines 4 and 9 can be unmodified, mono-, di-, or trimethylated or acetylated. Threonines 3 and 6 as well as serine 10 can be unmodified or phosphorylated. Peptides are tethered to a solid-support via a linker comprised of methionine, arginine and a PEG spacer. (b) A digital image of the library screen for the JMJD2A double tudor domain. Dark blue beads are marked with a circle (○) and colorless beads with a rectangle (□).

Figure 2

Figure 2. Graphical depiction of discrimination factors obtained from H3 library screens

Values for discrimination factors were obtained by dividing the percent frequency of each modification observed in the intensely blue pool for a given screen by the percent frequency of each corresponding modification from a random group of 100 library members. Discrimination factors represent the fold-likelihood of observing a particular modification in a protein screening experiment relative to random chance. Chi-squared values for each residue are reported along the x-axis. Serine and threonine residues allow for 1 degree of freedom (DF) while lysine and arginine allow for 4 DF. Values above the 99% confidence level for statistical significance are marked with an asterisk (*) (for 1 degree of freedom (DF) = 6.63 and 4 DF = 13.28). A double asterisk (**) is used to denote positions with notably high chi-squared values (above 99.9% confidence level for statistical significance where 1 DF = 10.83 and 4 DF = 18.47).

Figure 3

Figure 3. Detection of H3T6ph by Western blot analysis

(a) Spot blot Western with six distinct H3 peptides residues 1-11 (1:H3T3ph; 2:H3unmod; 3:H3T11ph; 4:H3T6ph; 5:H3K4me3T6ph; 6:H3S10ph). (b) H3T6ph-antibody recognizes H3T6ph from native histones extracted from HeLa cell nuclei (left panel). (c) A 1 μM H3T6ph (1-11) peptide competition diminishes the signal to background levels. The black arrow indicates the band corresponding to H3. The asterisk (*) most likely pertains to H3 C-terminal degradation products.

Figure 4

Figure 4. Detection of H3T6ph using mass spectrometry

(a) Extracted ion chromatogram of the [M+2H]2+ ion at 455.724 m/z, the expected mass of both H3T3phos and H3T6phos from HeLa cells. This sample was subjected to propionic anhydride derivatization, methyl esterification and immobilized metal affinity chromatography (IMAC) for facilitated analysis and phosphopeptide enrichment. As can be see, a major peak is observed at 16.1 minutes, while a second minor resolved peak is clearly visible at 15.8 minutes. Mass accuracy was found to be ~2ppm on either peak as recorded on an Orbitrap mass spectrometer. (b) MS/MS spectrum of the species eluting at 16.1 minutes. The MS/MS fragments show that the sequence is from the 3-8 residues of histone H3 containing T3 phosphorylation. (c) MS/MS spectrum of the species eluting at 15.8 minutes. The MS/MS fragments show that the sequence is from the 3–8 residues of histone H3 containing T6 phosphorylation. Note pr = propionyl amide (56 Da), phos = phosphorylation (80 Da), and —OMe = methyl ester (14 Da).

Figure 5

Figure 5. Identification of the H3K4me3T6ph-binding site of the ING2 PHD finger

The histograms show normalized 1H,15N chemical shift changes in backbone amides of the ING2 PHD finger induced by the H3K4me3T6ph (a) and H3K4me3 (b) peptides. The protein:peptide ratio is 1:5, which represents saturation for both interactions. (c) Superimposed 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the ligand-free (black), H3K4me3T6ph-bound (purple) and H3K4me3-bound (green) ING2 PHD finger (0.2 mM). (d) Residues that show a unique pattern of chemical shift perturbations in (c) are colored in red on the surface of the ING2 PHD-H3K4me3 complex and labeled. The residues that exhibit large but parallel chemical shift changes upon addition of either H3K4me3T6ph or H3K4me3 are colored in gray.

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