Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1 - PubMed (original) (raw)

. 2012 Aug 7;109(32):12950-5.

doi: 10.1073/pnas.1203701109. Epub 2012 Jul 25.

Shin Isogai, Takashi Oda, Motoko Unoki, Kazuya Sugita, Naotaka Sekiyama, Keiko Kuwata, Ryuji Hamamoto, Hidehito Tochio, Mamoru Sato, Mariko Ariyoshi, Masahiro Shirakawa

Affiliations

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

Kyohei Arita et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Structure of TTD-PHD of UHRF1 in complex with H3-K9me3. (A) Diagram of UHRF1 domains. (B) Structure of TTD-PHD bound to H3-K9me3. (Left and Right) Surface model (Left) and illustration model (Right) of TTD-PHD. Each region of TTD-PHD is colored according to the diagram in A. Histone H3 is shown as a green sphere; zinc ions are shown as gray spheres. (C) SAXS analyses are shown for wild-type TTD-PHD (green), TTD-PHDR295A/R296A (orange), and TTD-PHDS298ph (red).

Fig. 2.

Fig. 2.

Recognition of histone H3-K9me3 by UHRF1. (A) Electrostatic surface potential of TTD-PHD. Surface colors represent the potential from −15 KBT−1 (red) to 15 KBT−1 (blue). Histone H3-K9me3 is shown by a ball-and-stick model. (Right) Close-up view of H31–10, fitting into the hole of TTD-PHD. (B) Recognition of the cassette 1 region of histone H3 by TTD-PHD. Selected residues involved in the TTD-PHD:H3 interfaces are indicated. The color coding is the same as in Fig. 1_A_. Red dotted lines indicate hydrogen bonds. The 2|_F_o| − |_F_c| difference Fourier map for histone H3 (>1.0 σ) is shown in blue. (C) Recognition of the cassette 2 region of the histone H3 tail. The residues of TTD-PHD involved in histone H3 recognition are shown as a cyan stick model, with nitrogen and oxygen atoms in blue and red, respectively.

Fig. 3.

Fig. 3.

Structure of the histone H3 tail. (A) Overlay of the 1H-15N correlation spectra of the histone H3 tail (residues 1–20, containing the K9me3 analog) alone and bound to wild-type TTD-PHD in red and blue, respectively. Structure of the H3-K9me3 peptide superimposed on the 2|_F_o| − |_F_c| difference Fourier map contoured at 1.0 σ (blue) is depicted in the box. (B) Chemical shift deviations of 13CA and 13CO from the random coil values in the histone H3-K9me3 peptide.

Fig. 4.

Fig. 4.

Disruption of the linker:Tudor contacts alters the binding mode of TTD-PHD to the histone H3 tail. (A) Contacts between the linker and TTD. The side chains of the linker residues are shown as ball-and-stick models in magenta. The space-filling model in yellow indicates S298. (Upper Right and Lower Right) Close-up views of the intermodule junction (Upper Right) and the interactions (Lower Right) between the linker and TTD. (B) ITC measurements of wild-type TTD-PHD, TTD-PHDR295A/R296A, TTD-PHDS298ph, and an equimolar mixture of isolated TTD and PHD finger with H3-K9me3 peptide. The integrated heat of each injection is depicted. The ITC thermograms of all experiments are shown in

SI Appendix, Figs. S1–S3

. (C) Phosphorylation of TTD-PHD S298 in E. coli. Phosphorylation by rPKA was confirmed by a gel shift-mobility assay using phos-tag SDS/PAGE. The total amount of protein in each lane was 0.3 μg. AK indicates alkaline phosphatase treatment to remove a phosphate group. (D) 1H-15N correlation spectrum of H3-K9me3 peptide alone (red) overlaid with that in the presence of a 2-molar excess of TTD-PHDR295A/R296A, TTD-PHDS298ph, or an equimolar mixture of the isolated TTD and PHD finger (blue). The dotted circle shows the peaks of residues T6-R8 of H3, which are representative peaks of helical structure in the complex with wild-type TTD-PHD.

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