Histone H3Q5 serotonylation stabilizes H3K4 methylation and potentiates its readout

Abstract

Serotonylation of glutamine 5 on histone H3 (H3Q5ser) was recently identified as a permissive posttranslational modification that coexists with adjacent lysine 4 trimethylation (H3K4me3). While the resulting dual modification, H3K4me3Q5ser, is enriched at regions of active gene expression in serotonergic neurons, the molecular outcome underlying H3K4me3-H3Q5ser crosstalk remains largely unexplored. Herein, we examine the impact of H3Q5ser on the readers, writers, and erasers of H3K4me3. All tested H3K4me3 readers retain binding to the H3K4me3Q5ser dual modification. Of note, the PHD finger of TAF3 favors H3K4me3Q5ser, and this binding preference is dependent on the Q5ser modification regardless of H3K4 methylation states. While the activity of the H3K4 methyltransferase, MLL1, is unaffected by H3Q5ser, the corresponding H3K4me3/2 erasers, KDM5B/C and LSD1, are profoundly inhibited by the presence of the mark. Collectively, this work suggests that adjacent H3Q5ser potentiates H3K4me3 function by either stabilizing H3K4me3 from dynamic turnover or enhancing its physical readout by downstream effectors, thereby potentially providing a mechanism for fine-tuning critical gene expression programs.

Keywords: H3K4me3; H3Q5 serotonylation; designer chromatin; histone modification; modification crosstalk.

Fig. 1.

Profiling of H3K4me3 readers in response to H3K4me3Q5ser dual modification. (A) Structure of the Q5ser modification. Carbon atoms are shown in yellow; nitrogen atoms are shown in blue; oxygen atoms are shown in red. (B) The coexistence mode of the H3K4me3Q5ser dual modification. (C) SPRi signal of H3(1–15)K4me3 and H3(1–15)K4me3Q5ser peptides toward H3K4me3 readers. The values of SPRi signals are the blanked average signal during 250–300 s of the association curve in SI Appendix, Fig. S1A. (D) Titration curves and fitting curves of H3(1–15)K4me3 and H3(1–15)K4me3Q5ser peptides titrated into H3K4me3 readers. K_D values are provided (see SI Appendix, Table. S1 for fitting parameters and error). (E) Summary of binding affinities determined in D. (F) ITC curves of TAF3_PHD binding to histone H3 unmodified vs. Q5ser, K4me1 vs. K4me1Q5ser, K4me2 vs. K4me2Q5ser, and K4me3 vs. K4me3Q5ser peptides. K_D values are provided (see SI Appendix, Table S1 for fitting parameters and error). (G) ITC curves of TAF3_PHD binding to histone H3K4me3Q5(ser-analog) peptides. K_D values are provided (see SI Appendix, Table S1 for fitting parameters and error).

Fig. 2.

Influence of the H3Q5ser modification on the MLL1 complex, an H3K4me3 writer. (A) Kinetic model of two irreversible consecutive reactions catalyzed by the MLL1 complex. k₁ and k₂ represent the pseudo–first-order rate constants for the conversion of K4me0→K4me1 and K4me1→K4me2, respectively. (B) MALDI-TOF mass spectrometry of MLL1 complex enzymatic assays using H3(1–15)K4me3 and H3(1–15)K4me3Q5ser peptides as substrates. (C) Kinetic fitting of methylation reactions on H3(1–15)un and H3(1–15)Q5ser substrates determined by LC-MS. Summary of rate constants (k₁ and k₂) derived from globally fitting experimental data are labeled in the panel. (D) The catalytic center of MLL1 in complex with the H3K4 peptide (coordinates were taken from the Protein Data Bank [PDB] entry 2W5Z). H3K4 and Q5 residues are shown as yellow sticks; the binding surface of MLL1 is shown as a white surface. Q5ser modification is modeled as cyan sticks. The H3Q5ser residue stretches out of the MLL1-binding surface.

Fig. 3.

Impact of H3Q5ser modification on LSD1 and KDM5B mediated demethylation of H3K4. (A) MALDI-TOF demethylation assay using H3(1–25)K4me3 vs. H3(1–25)K4me3Q5ser peptide substrates (Top) and H3(1–25)K4me2 vs. H3(1–25)K4me2Q5ser peptide substrates (Bottom). Demethylation of H3(1–25)K4me3 resulting in the H3(1–25)K4me1/2 product is visible after 4 h, while no demethylation is observed for H3(1–25)K4me3Q5ser. *Impurity from peptide synthesis. †Residual K4me1Q5ser species from synthesis of K4me2Q5ser substrates, unable to purify. (B) MALDI-TOF demethylation assay as described in A using recombinant LSD1. LSD1 robustly demethylates native substrates K4me2 (Top) and K4me1 (Bottom), while no demethylation activity is seen for substrates containing Q5ser. (C) Western blot of 60-min time course of KDM5B demethylation of 12-mer chromatin array substrates. In the presence of KDM5B, H3K4me3 arrays are readily demethylated, while no demethylation occurs for H3K4me3Q5ser-containing arrays. (D) Western blot of KDM5B concentration course. Increased amounts of KDM5B do not promote demethylation of H3K4me3Q5ser arrays. (E) Increasing 12-mer array substrates in the KDM5B demethylation assay does not promote demethylation of H3K4me3Q5ser arrays.

Fig. 4.

KDM5B inhibition of demethylation of semisynthetic 12-mer chromatin arrays. (A) Schematic of mixed 12-mer array substrates. Both H3 tails are modified. (B) H3K4me0 on a neighboring mononucleosome stimulates KDM5B demethylation activity of H3K4me3 but not H3K4me3Q5ser. (C) H3Q5ser on a neighboring mononucleosome does not inhibit stimulation of KDM5B by KDM5B_PHD1. (D) Normalized fluorescence polarization of GST-KDM5B_PHD1 binding to H3 unmodified (blue) and H3Q5ser (red) peptides. N-terminally acetylated H3 was included as a negative control (black). Errors represent ± SD of n = 3. (E) Experimental flow for chromatin dipping in active, isolated HeLa lysate. (F) Chromatin array dipping in isolated HeLa nuclear lysate followed by streptavidin enrichment shows dramatically less demethylation of H3K4me3Q5ser arrays compared to H3K4me3. Representative Western blot shown from four replicate experiments. t test (two-tailed), 95% confidence; ****, < 0.0001. Errors represent ± SD (G) The catalytic center of JMJ14 (the H3K4me3 eraser in Arabidopsis thaliana) in complex with an H3K4me3 peptide (coordinates were taken from the PDB entry 5YKO). H3K4 and Q5 residues are shown as yellow sticks; the binding surface of JMJ14 is shown as a white surface. The hydrogen bond between H3Q5 and D312 is highlighted with a red dash. Q5ser modification is modeled as cyan sticks. H3Q5ser causes steric clash at the catalytic center of JMJ14. (H) The catalytic center of LSD1 in complex with the histone H3 peptide (coordinates were taken from the PDB entry 2V1D). H3M4 (K4me3 mimic) and Q5 residues are shown as yellow sticks; the binding surface of LSD1 is shown as a white surface. H3Q5ser causes steric clash at the catalytic center of LSD1. (I) Model representing the different degrees of crosstalk between H3K4me3 and H3Q5ser. KDM5/LSD1 are inhibited by Q5ser, MLL1 remains active against Q5ser-modified substrates, albeit with a slower k₂, and the reader TAF3 displays increased binding to H3K4me3Q5ser.