PHD Finger Recognition of Unmodified Histone H3R2 Links UHRF1 to Regulation of Euchromatic Gene Expression

Abstract

SUMMARY Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHDUHRF1), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

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.…”

“…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).…”

“…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.…”

“…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).…”

DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV

“…S6C), a domain family that is similar to the classic chromo domain in sequence but adopts a β-barrel instead of the α + β fold of the chromo domain (27). A subset of the proteins containing chromo barrel or chromo domains has been shown to bind methylated histones and the interaction involves 3-4 conserved aromatic residues that form an "aromatic-cage" to pocket the methylated lysine from histones (28)(29)(30). Interestingly, the three aromatic residues are also conserved in the N-terminal half of the SAWADEE domains of DTF1 and DTF2 (Fig.…”