Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

Abstract

We report two NMR complex structures of PHD UHRF1 binding to unmodified or K9 trimethylated histone tails, which clarify a controversy regarding how the binding of UHRF1 to H3 tails is mediated. Based on our structures, H3R2, not H3K9, mediates PHD binding.Human UHRF1, also known as ICBP90, plays central roles in maintaining CpG DNA methylation [1,2] and epigenetic code inheritance [3]. It harbors five recognizable functional domains: an ubiquitin-like domain (UBL) at the N-terminus, a tandem tudor domain (Tudor UHRF1 ), a plant homeodomain (PHD UHRF1 ), a SET and RING associated (SRA) domain and a RING domain at the C-terminus, which mediate interactions of UHRF1 with histones and DNA ( Figure 1A). Previous studies suggested that PHD UHRF1 defines binding specificity of UHRF1 to trimethylated H3 tail H3K9me3 [3]. However, recent studies showed that H3K9me3 binding is mediated by the Tudor UHRF1 [4,5]. PHD UHRF1 is structurally different from the canonical PHD fingers (Figure 1). It has additional Cys residues at its amino terminal portion; it is also devoid of the residues to form the H3K4me3-binding aromatic cage [6][7][8][9] and a terminal aspartic acid residue to recognize H3K4me0 [10][11][12][13]. Therefore, exactly how PH-D UHRF1 binds to the N-terminal H3 tail remains unclear.To address this issue, we determined 3D NMR structures of free PHD UHRF1 , and two complexes, namely PH-D UHRF1 -H3K9me0 and PHD UHRF1 -H3K9me3 (Figure 1 and Supplementary information, Data S1, Figure S1, Table S2). In these three structures, PHD UHRF1 is coordinated by three zinc ions to form a rod-shape structure, containing a small α-helix, a double-stranded anti-parallel β-sheet and three loops. The free and bound PHD UHRF1 structures have backbone atoms RMSD value of 1.7 Å, indicating that H3 peptides binding does not induce overall major conformational changes of PHD UHRF1 . The C-terminus of PHD UHRF1 is coordinated by two zinc ions in an interleaved, compact manner, which is similar to the reported histone tail-binding PHD domain structures (Figure 1) [6][7][8][9][10][11][12][13]. However, a difference lies at the N-terminus of In both structures of PHD UHRF1 -H3K9me0 and PH-D UHRF1 -H3K9me3 ( Figure 1B-1E), the N terminus of H3 peptide is anchored through nonpolar interactions between methyl group of H3A1 and hydrophobic side chains of PHD UHRF1 residues L344, P366 and W371 and hydrogen bonds between H3A1 nitrogen atom and backbone oxygen of PHD UHRF1 P366 and D369. Apparently, H3 residues R8 and K9 (unmethylated or trimethylated) have no interaction with PHD UHRF1 , with their sidechains extending outwards. Recently, it was reported that H3K9me3 is recognized via tandem Tudor domain [4,5], this may explain why the side chain of H3K9 does not make contact with PHD UHRF1 in our complex structures. The first zinc finger almost has no contact with histone peptides ( Figure 1B-1E), except that backbone oxygen atom of PHD UHRF1 C329 forms a hydrogen bond with the side-chain nitrogen atom (Nξ) of H3K4. The second zinc finger seem...

Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1

“…1A). In agreement with previous reports (19,20,23,25), our binding assay using isothermal titration calorimetry (ITC) showed that isolated TTD and PHD bound to the N-terminal H3 peptide depending on K9me3 and unmodified R2, respectively (Table 1 and SI Appendix, SI Results). We also observed 1:1 stoichiometric binding of TTD-PHD to the H3 tail bearing unmodified R2 and K9me3 with significantly higher affinity (K d = 0.37 μM) compared with TTD (K d = 1.75 μM) or PHD finger (K d = 1.47 μM) alone (Table 1 and SI Appendix, Fig.…”

“…Trimethylation of histone H3K9 is well known to be associated with transcriptional suppression (31), but the role of H3-R2 methylation has just started to be unveiled (32)(33)(34)(35). Recently, H3-R2 binding of UHRF1 was shown to be dispensable for localization of UHRF1 at pericentromeric heterochromatin (19,20). However, the biological relevance of the histone code (H3-R2me0-K9me3) that is recognized by the TTD-PHD of UHRF1 remains to be clarified.…”

“…2B and SI Appendix, Fig. S5B) (19,20). Residues 2-4 of H3 cassette 1 make main-chain hydrogen bonds to form an intermolecular β-sheet with the PHD finger (residues 330-333) ( methylation of H3-R2 decreased the binding affinity of the isolated PHD finger for the unmodified H3 peptide (K d = 12.70 μM) and also interfered with the interaction of H3 with TTD-PHD regardless of H3-K9 methylation (Table 1).…”

“…The histone H3 peptides H3 1-10 and H3 1-12 -K9me3 for crystallography and a series of H3 peptides for ITC measurements were purchased from Toray Research Center. Histones H3 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and H4 peptides harboring a site-and degree-specific methylated lysine analog were prepared by cysteine alkylation as reported previously (36, 37).…”

“…1A). The isolated PHD finger and TTD recognize the methylation states of Arg-2 (H3-R2) and Lys-9 (H3-K9) in the H3 tail, respectively (19)(20)(21)(22)(23). Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics analysis indicated that UHRF1 binds to nucleosomes containing trimethylated H3-K9 (H3-K9me3) (24).…”