Molecular basis for the methylation specificity of ATXR5 for histone H3

Abstract

In plants, the histone H3.1 lysine 27 (H3K27) mono-methyltransferases ARABIDOPSIS TRITHORAX RELATED PROTEIN 5 and 6 (ATXR5/6) regulate heterochromatic DNA replication and genome stability. Our initial studies showed that ATXR5/6 discriminate between histone H3 variants and preferentially methylate K27 on H3.1. In this study, we report three regulatory mechanisms contributing to the specificity of ATXR5/6. First, we show that ATXR5 preferentially methylates the R/F-K*-S/C-G/A-P/C motif with striking preference for hydrophobic and aromatic residues in positions flanking this core of five amino acids. Second, we demonstrate that post-transcriptional modifications of residues neighboring K27 that are typically associated with actively transcribed chromatin are detrimental to ATXR5 activity. Third, we show that ATXR5 PHD domain employs a narrow binding pocket to selectively recognize unmethylated K4 of histone H3. Finally, we demonstrate that deletion or mutation of the PHD domain reduces the catalytic efficiency (kcat/Km of AdoMet) of ATXR5 up to 58-fold, highlighting the multifunctional nature of ATXR5 PHD domain. Overall, our results suggest that several molecular determinants regulate ATXR5/6 methyltransferase activity and epigenetic inheritance of H3.1 K27me1 mark in plants.

Figure 1.

ATXR5 recognizes an extensive motif on H3.1. (A) Surface representation of the ATXR5 crystal structure bound to histone H3.1 (revised model of 4o30). The surface of the protein is highlighted in dark gray and the peptide is colored orange. (B) Zoomed view representation of ATXR5 binding cleft. ATXR5 and histone H3 residues are rendered in gray and orange residues, respectively. Carbon and oxygen atoms are colored in red and blue respectively. Hydrogen bonds are shown in red dash lines. For clarity, only a subset of the interactions are shown (C) Heat map representation of average methylation intensities measured by phosphorimaging shown in Supplementary Figure S1. Color code representing the degree of methylation: white: same methylation intensity as wild type; blue: loss of methylation intensity; red: gain of methylation intensity. (D) Sequence logo of the relative methylation factor for every amino acid at each position of the peptide arrays (n = 3) performed with the program Seq2logo (http://www.cbs.dtu.dk/biotools/Seq2Logo/), using the PSSM-logo method. Amino acids are sorted from top to bottom in order of importance. Enriched amino acids are represented on the positive y-axis and depleted amino acids are shown in the negative y-axis. Color code: blue: positively charged; red: negatively charged; green: polar; black: all other amino acids.

Figure 2.

Post-translational modifications of residues neighboring histone H3.1 K27 are detrimental to ATXR5 enzymatic activity. (A) Schematic representation of the H3.1 peptide showing the PTMs deposited in vicinity of the K27 methylation site (red). (B) Methyltransferase activity of ATXR5 SET domain on modified histone H3.1 peptides relative to un-modified H3.1. (C) Zoomed-view of ATXR5 SET domain binding cleft showing un-modified S28 (left) in which the carbon atoms of the peptides and proteins are colored in orange and green respectively. (D) Comparison of ATXR5/H3.1/AdoHcy with ATXR5/H3.1K36me3/AdoHcy in which the carbon atoms for the modified peptides and co-crystallized ATXR5 are colored in yellow and white, respectively. Zoomed-view of ATXR5 SET domain binding cleft showing un-modified R26 (E), R26me1 (F) and R26me2a (G). Carbon atoms of the un-modified and modified peptides are rendered in orange and yellow respectively. Hydrogen bonds are highlighted by red dashed lines.

Figure 3.

Structure of ATXR5 PHD domain in complex with H3K4 peptide. (A) ATXR5 PHD domain is shown in ribbon representation and colored teal. Secondary structure elements (α helices, β strands and loops) are indicated. The N and C termini of the PHD domain are also indicated. Zn atoms are shown as red spheres. The H3K4 peptide is shown in stick representation with carbon atoms colored yellow, oxygen atoms red, nitrogen atoms blue. Figure was rendered with PyMOL (http://pymol.org). (B) Molecular surface representation of the ATXR5 PHD domain colored according to electrostatic potential (blue, positive [+10 kT/e]; white, neutral; red, negative [−10 kT/e]). The H3K4 peptide is shown in stick representation and coloring scheme is the same as in (A). Inlet figures show a zoomed view of the residues interacting with K4 (left panel) and with A1 and R2 (right panel). The electrostatic surface representation was made using the Adaptive Poisson Boltzmann Equation (APBS) plugin in PyMOL (44).

Figure 4.

ATXR5 activity is stronger on the NCP and with an intact PHD domain. KMTase assays were performed using histone octamers or NCP as substrate, using tritiated S-adenosyl-l-Methionine and varying amounts of WT or mutant enzyme. (A) Reactions performed with ATXR5 WT or a mutant in which the PHD domain has been deleted. Reaction products were separated by SDS-PAGE, gel extracted using isopropanol and radioactivity on histones was quantitated by scintillography. (B) Similar reactions were performed, this time comparing ATXR5 WT or L39W mutant. Reactions were stopped by spotting the reactions on P81 filter paper and quantified by scintillography.

Figure 5.

Cofactor steady-state kinetics are affected by the mutation of ATXR5 PHD domain. Michaelis–Menten (M–M) plot of the initial velocity versus nucleosome concentration (A) and its Lineweaver–Burk (LB) double reciprocal plot (B) for full-length wild-type ATXR5, L39W mutant and ATXR5 ΔPHD. The kinetics for the cofactor were performed using 8 μM of recombinant nucleosome. The M-M plot of the initial velocity versus cofactor concentration (C) and its corresponding LB plot (D) for the constructs used in C. (E) Summary of the results obtained from the M-M plots for wild-type ATXR5, L39W mutant and ATXR5 ΔPHD.

Figure 6.

Increasing cofactor concentration bypasses the inability of ATXR5 mutant to bind the nucleosome. EMSA experiments were performed by incubating increasing amounts of ATXR5 WT in presence of 10X (A) or 1000X (C) of sinefugin (SFG) and fixed amounts of fluorescently-labeled NCP. Similar EMSAs were performed with ATXR5 L39W in 10X (B) or 1000X (D). Reactions were resolved on non-denaturing polyacrylamide gels. The * indicates the ATXR5–NCP complexes and the protein concentrations are indicated on the top of each gel. (E) GST pull-down assays showing the interaction between ATXR5 PHD domain with untagged ATXR5 SET domain in presence of sinefungin. Proteins were resolved on a denaturating SDS-PAGE gel and stained with Coomassie Brilliant Blue. (F) Model of the regulatory elements controlling the activity of ATXR5. The SET and PHD domains of ATXR5 are colored in green and blue respectively and are shown in two different conformations on active (top) or silent (bottom) chromatin and the cofactor is represented by a yellow circle. Histone tails of H3.3 and H3.1 are shown as black lines and the residues with their respective PTMs are indicated.