Molecular determinants for recognition of serotonylated chromatin

Abstract

Post-translational modifications of histone tails constitute a key epigenetic mechanism controlling chromatin environment and gene transcription. Serotonylation of histone H3Q5 (H3Q5ser) is a recently discovered mark associated with active transcription of RNA polymerase II (pol II)-transcribed genes. The direct link between H3Q5ser and the pol II transcription machinery relies on the TFIID subunit TAF3. The presence of H3Q5ser enhances TAF3 binding to H3K4me3, but the molecular determinants underlying this interaction remained unclear. Here, we resolve the binding mode of TAF3-PHD to H3K4me3Q5ser identifying a novel binding surface for H3Q5ser using solution nuclear magnetic resonance spectroscopy. This reveals how H3Q5ser recognizes a conserved surface of the TAF3-PHD via CH-π interactions in an edge-face conformation involving a proline residue stabilized by a tryptophan. This combination of proline and tryptophan is unique to the PHD finger of TAF3 and conserved among TAF3 orthologues. Our findings establish a framework for the molecular recognition of serotonylated chromatin, laying the foundation for developing epigenetic inhibitors targeting serotonylation-dependent transcriptional regulation in neuronal development.

Graphical Abstract

Figure 1.

H3Q5 serotonylation enhances TFIID binding in all H3K4 methylation states. Quantitative mass spectrometry (qMS) confirms that TFIID has a preference towards H3Q5ser peptides in (A) H3K4me0, (B) H3K4me1, (C) H3K4me2, and (D) H3K4me3. False discovery rate cut-off is 0.1. Colour coding of identified proteins is indicated in the centre of the figure.

Figure 2.

NMR analysis of Taf3-PHD finger bound to H3K4me peptides with and without H3Q5ser. (A) Overlay of 2D ¹⁵N-HSQC (left) and ¹³C-HSQC (proline Cδ–Hδ region, bottom right) spectra of Taf3-PHD with excess of indicated peptides. Resonances with significant CSPs are labelled and connected by dashed lines. Residues with H3Q5ser-dependent chemical shifts are indicated with magenta labels, with A902 and S880 highlighted and expanded in the boxes. (B) Structure of Taf3-PHD bound to H3K4me3 peptide (in green) (PDB 2K17) colour coded with CSPs between average chemical shift of the non-serotonylated complexes (H3K4me3/H4K4me2/H3K4me1) and the corresponding Q5 serotonylated complexes. (C) Normalized peak intensities of non-overlapping Taf3-PHD residues in complex with H3K4me3Q5ser and H3K4me2Q5ser peptides, relative to their non-serotonylated counterparts. (D) As the panel (B), but here colour coded according to the normalized peak intensity for the H3K4me3Q5ser complex, relative to the H3K4me3 complex. Selected residues are labelled. Colour coding indicated in the figure.

Figure 3.

H3Q5ser stabilizes Taf3-PHD/H3K4me2 and Taf3-PHD/H3K4me3 complexes. (A) Experimental (blue) and fitted (red) spectra of free, ∼50% bound, and fully bound TAF3-PHD resonances E862 and C896 for H3K4me3/H3K4me3Q5ser complex. Transfer peaks indicated with *. Experimental spectrum of E862 is also shown separately for clarity. (B) ITC analysis of GST-tagged Taf3-PHD binding to H3K4me3 in yellow and H3K4me3Q5ser in navy blue. Top panel displays the raw heat change per injection of H3 peptide. Bottom panel represents the binding isotherm. The solid line represents the best fit to the one-set-of-sites model using MicroCal-ITC Origin 7 software.

Figure 4.

Solution NMR structure of the Taf3-PHD/H3K4me3Q5ser complex. (A) Most representative structure of the complex in cartoon depiction. Coordinated zinc atoms shown as spheres, with coordinating residues in sticks. (B) Close-up on the binding interface with intermolecular hydrogen bonds conserved across the ensemble shown as black dashed lines. (C) Superposition of the ensemble of 20 solution structures, zoomed in on the Q5ser binding site. (D) Close-up on the Q5ser binding site showing the molecular surfaces of Taf3-PHD and H3K4me3Q5ser to illustrate the stacking of H3Q5ser on P881 and W894.

Figure 5.

Taf3-PHD P881 and W894 form key interaction surface with the serotonin group. (A) ITC analysis of GST-tagged Taf3-PHD mutants, BPTF-PHD [2], PHF2-PHD, or PHF8-PHD with the peptides H3K4me3 (in yellow) and H3K4me3Q5ser (in blue). Top panels display the raw heat change per injection of H3 peptide. Bottom panels represent the binding isotherm. The solid line represents the best fit to the one-set-of-sites model using MicroCal-ITC Origin 7 software. (B) Multiple sequence alignment (MSA) of human PHD fingers for which high-resolution structures in complex with H3K4me3 are available (ASH1L PDB: 8VLF; BPTF(2) PDB: 2FUU; DIDO1 PDB: 4L7X; ING1 PDB: 2QIC; ING4 PDB: 2PNX/2VNF; ING5 PDB: 3C6W; KMT2E PDB: 4L58; PHF2 PDB: 3KQI; PHF13 PDB: 3O7A; UHRF1 PDB: 3SOW). Consensus Cys4–His–Cys3 positions are highlighted in grey and indicated on top. Locations of the secondary structure elements of TAF3-PHD are indicated on top. S880 and A902 are highlighted in purple, while P881 and W894 are highlighted in yellow.

Figure 6.

Observed and modelled edge–face interactions with H3Q5 monoaminylation modifications. (A) Comparison of H3Q5ser edge–face interaction with Taf3-PHD in panel (B) and WDR5 (PDB: 7CFP) in panel (C). Structure of H3Q5ser (D) compared to model of H3Q5his (E) and H3Q5dop (F) in complex with Taf3-PHD. Atomic coordinates for histamine and dopamine ligands (from the PDB) were superimposed on the serotonin aromatic in the H3K4me3Q5ser/Taf3-PHD complex. In this conformation, the histamine and serotonin rings stack exclusively on P881. Stacking on W894 would require conformational changes in the Q5 side chain and the his/dop linker to shift the position of the ring.