The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Abstract

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.

Figure 1.

Solution structure of AIRE-PHD1 in complex with H3K4me0. (A) Stereoview of the ensemble of 20 complex structures, the backbone traces of AIRE-PHD1 and the side chains of the peptide are represented in pink and blue, respectively. (B) Cartoon representation and (C) surface plot of the lowest energy complex structure. Cyan-dashed lines represent intermolecular hydrogen bonds. Colour coding: pink, AIRE-PHD1; blue, H3K4me0; orange, protein residues forming specific polar contacts with H3K4me0; green, protein residues forming hydrophobic contacts with H3K3me0; grey, Zn²⁺ ions.

Figure 2.

Structural comparison with other PHD:H3 complexes. Cartoon representations of (A) AIRE-PHD1:H3K4me0, (B) DNMT3L-Cys-rich:H3K4me0 (2PVC), (C) BHC80-PHD:H3K4me0 (2PUY), (D) Taf3-PHD:H3K4me3 (2K17), (E) Pygo-BCL9-PHD:H3R2me2asK4me2 (2VPG) and (F) RAG2-PHD:H3R2me2sK4me3 (2V87). The side chains of R2 and K4 (blue) and their specific interactions with the different PHD domains are explicitly shown. Intermolecular hydrogen bonds are represented in blue with dotted lines.

Figure 3.

Effect of H3 post-translational modifications on binding to AIRE-PHD1. (A) Tryptophan fluorescence binding curves of AIRE-PHD1 with H3K4me0, H3R2me2sK4me0 and H3R2me2asK4me0. (B) Interaction between GST-AIRE-PHD1 fusion protein and amino-terminal histone H3 peptides, all detected by anti-GST antibody. (C) ITC-binding curves for H3K4me0, H3R2me1K4me0 and H3T3PhK4me0 to AIRE–PHD1. The upper panels show the sequential heat pulses for peptide–protein binding, and the lower panels show the integrated data, corrected for heat of dilution and fit to a single-site-binding model using a nonlinear least-squares method (line). (D) Distribution of the backbone amide chemical shift changes observed in ¹⁵N-labelled AIRE–PHD1 (0.2 mM) upon addition of a threefold excess of H3K4me0, H3R2me1K4me0, H3R2me2sK4me0 and H3R2me2asK4me0. The asterisks indicate residues whose backbone amide signals disappear during titration owing to line broadening. H3R2me1K4me0 (H3R2me2sK4me0, H3R2me2asK4me0), histone H3 monomethylated (respectively, symmetrically and asymmetrically dimethylated) at arginine 2. Tryptophan fluorescence-binding curves, ITC-binding curves and NMR titrations of AIRE-PHD1 with H3K4me0 refer to previous experiments reported in (22).

Figure 4.

R2 methylation hinders AIRE ability to activate target genes. (A) Western blot analysis of transfected HEK293 cells. Transfections were carried out as indicated below, antibodies and/or detected proteins are indicated on the right. (B) Relative expression level of AIRE regulated involucrin (INV) and S100A8 genes and AIRE independent S100A10 gene. Activations, measured with quantitative RT–PCR, are shown as relative quantity of mRNA compared to control transfection (= 1) of each mRNA. The data are the averages of two independent transfections.