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. 2008 Aug 5;47(31):8094-102.
doi: 10.1021/bi800766k. Epub 2008 Jul 11.

A combinatorial H4 tail library for exploring the histone code

Adam L Garske  1 , Gheorghe Craciun, John M Denu
Affiliations

A combinatorial H4 tail library for exploring the histone code

Adam L Garske et al. Biochemistry. .

Abstract

Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first 21 residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59 of 60 sequences were phosphorylated at S1, while 30 of 30 of those selected from the nonhits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least-squares fitting of modifications from the identified peptides (40 hits and 34 nonhits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation and acetylation of neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with 13 H4 peptides. Dissociation constants ranged from 1 mM to 1 microM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein.

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Figures

Figure 1
Figure 1
The H4 histone tail library corresponds to the first 21 amino acids of human histone H4 and is attached to a linker composed of two β-alanines (B) and a methionine. Citrulline is demarcated by U and N-termini are acetylated (Ac). Sites of variation are above/below X’s.
Figure 2
Figure 2
Generalized on-bead assay scheme. To determine histone code reader (i.e. histone binding protein) specificity, the biotinylated protein is incubated with the library prior to addition of streptavidin-conjugated alkaline phosphatase, which recruits and turns over bromo-4-chloro-indolyl phosphate (BCIP) in the vicinity of beads bearing interacting peptide sequences. Turnover of BCIP leaves a blue precipitate on these beads.
Figure 3
Figure 3
Frequency of PTMs observed from the (a) intensely blue and (b) colorless populations of beads at amino acid positions 1, 3, 5, 8, 12, 16 and 20 when the combinatorial H4 tail library was screened with hJMJD2A DTD at 50 nM and 2 μM concentrations, respectively.
Figure 4
Figure 4
Histone code fingerprints (HCFs) depicting the combinatorial interplay of PTMs (at sites other than K20) observed for binding of the hJMJD2A DTD to histone H4 tails. The HCF’s are correlation matrices of the PTMs observed from a) sequences from intensely blue beads in the 50 nM screen b) sequences from colorless beads in the 2 μM screen. Numerical values for each square were determined by calculating the correlation coefficients for matrices consisting of each grouping of peptides (numerical assignments for each modification can be found in the main text). The color of each square signifies the strength and direction of the relationship between any two amino acid positions. Positive values on the colorbar represent correlations while negative values represent anti-correlations.
Figure 5
Figure 5
Sample plot of ITC of JMJD2A double tudor domain with 8. Raw titration data and integrated heats are shown in the top and bottom panels respectively. Different volumes were injected during the course of the experiment (1 × 1 μL, 6 × 4 μL and 33 × 8 μL injection). Dissociation constant (Kd = 3.5 ± 0.1 μM) and stoichiometry of binding (n = 0.9) were determined by Lavenberg-Marquardt nonlinear regression.
Figure 6
Figure 6
a) Representation of hJMJD2A DTD binding affinity with horizontal lines demarcating various modification states (binding differences not shown to scale). The hJMJD2A DTD: H4 interaction is primarily controlled by the methylation state of K20, but alterations in the net acetylation state result in incremental changes in affinity. b) Working model for JMJD2A-mediated transcriptional repression. Histone H4 monomethylation at K20 and hyperacetylation correlates with transcriptionally-poised chromatin while trimethylation of K20 and hypoacetylation tracks with transcriptional repression. The histone modification state is controlled by the dynamic action of HATs, HMTs (histone methyltransferases), HDACs and histone demethylases. The hJMJD2A DTD preferentially binds to transcriptionally-repressive H4 where it can recruit HDACs and potentially localize demethylase activity.
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