Shiraishi N et al. (2024), Structure of human DPPA3 bound to the UHRF1 PHD...

XB-ART-60757

Commun Biol 2024 Jun 19;71:746. doi: 10.1038/s42003-024-06434-9.

Show Gene links Show Anatomy links

Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3.

Shiraishi N , Konuma T , Chiba Y , Hokazono S , Nakamura N , Islam MH , Nakanishi M , Nishiyama A , Arita K .

???displayArticle.abstract???
DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique α-helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

???displayArticle.pubmedLink??? 38898124
???displayArticle.pmcLink??? PMC11187062
???displayArticle.link??? Commun Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: dnmt1 uhrf1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Characterization of the interaction between hUHRF1 and hDPPA3.a Amino acid sequence alignment of C-terminal part of DPPA3. Secondary structures of mouse and human DPPA3 are indicated based on PDB:7XGA and analysis of this study, respectively. b Schematic of the domain composition of human UHRF1 and DPPA3. c Isothermal titration calorimetry measurements for hPHD and wild-type (WT)/mutants of hDPPA381-118. Superimposition of enthalpy change plots with standard deviations. Data were presented as mean values for n = 3. d Overlay of 1H-15N heteronuclear single quantum coherence (HSQC) spectra of 30 µM hPHD showing chemical shift changes upon titration with hDPPA381-118 of 0 µM (black), 15 µM (blue), 30 µM (green), and 60 µM (red). Square regions inside the HSQC spectra were expanded (lower panels).
	Fig. 2. Crystal structure of hDPPA381-118 in complex with hPHD.a Overall structure of hPHD:hDPPA381-118 complex. Pre-PHD, core-PHD and hDPPA3 are depicted as gold, salmon, and cyan cartoon models, respectively. The conserved VRT motif in hDPPA3 is displayed as a stick model. Inset shows the interaction between the VRT motif of hDPPA3 and hPHD. The black dotted line represents a hydrogen bond. b Structural comparison of hPHD:hDPPA3 (this study, upper left), mPHD:mDPPA3 (PDB: 7XGA, upper right), hPHD:H3 (PDB: 3ASL, bottom left) and hPHD:PAF15 (PDB: 6IIW, bottom right) complexes. mDPPA3, H3 and PAF15 are shown as a green cartoon model and VRT (ART) motif are represented as stick model. c Electrostatic surface potential of hPHD calculated with program APBS56. The red and blue surface colors represent negative and positive charges, respectively. hDPPA3 is depicted as a cyan cartoon.
	Fig. 3. Solution structure of hDPPA3.a CD spectra of hPHD alone (red), hDPPA381-118 alone (blue), and the hPHD in complex with hDPPA381-118 (black). The sum of CD spectra of hPHD alone and hPDDA381-118 alone is shown as gray. b Dimensionless Kratky plots of hPHD alone (red diamond), hDPPA381-118 alone (blue square), and hPHD in complex with hDPPA381-118 (black circle) derived from small-angle X-ray scattering (SAXS) data. c Comparison of scattering curve derived from experimental data (cyan) and theoretical curve of the crystal structure of the hPHD:hDPPA381-118 complex (red). d Structural comparison of solution and crystal structures of the hPHD:hDPPA381-118 complex. Ab initio bead model of the hPHD:hDPPA381-118 complex derived from the SAXS scattering data (transparent gray sphere) is superimposed on the crystal structure (cartoon).
	Fig. 4. Competitive assay between hDPPA3 and the histone H3 tail.a Overlay of 1H-15N HSQC spectra of 15N-labeled hPHD in the presence of hDPPA381-118 and/or the H31-37W peptide at a molar ratio of 1:0:0 (black), 1:2:0 (green), 1:0:2 (blue), and 1:2:2 (red) of hPHD:hDPPA3:H3 (upper), and of 1:0:0 (black), 1:0:2 (blue), and 1:2:8 (yellow) of hPHD:hDPPA3:H3 (lower). b In vitro ubiquitination assay. C-terminal FLAG tagged-H31–37W was ubiquitinated using in-house purified E1, E2, and human UHRF1 (E3). The ubiquitinated H3 was detected using anti-FLAG antibody. Upper panel shows that 20, 40, and 100 µM hDPPA381-118 was added to the reaction solution including 20 µM of H31–37W. The lower panel presents results of an in vitro ubiquitination assay using 40 µM hDPPA381-118 mutants. The gel image is representative of n = 3 independent experiments.
	Fig. 5. Functional assay of DPPA3 using Xenopus egg extracts.a Experimental design for functional analysis of DPPA3 using Xenopus egg extracts. b Sperm chromatin was incubated with interphase Xenopus egg extracts supplemented with buffer (+buffer), 3×FLAG-mDPPA3, or 3×FLAG-hDPPA3. Chromatin fractions were isolated and immunoblotted using the indicated antibodies. The gel image is representative of n = 3 independent experiments. c Sperm chromatin was added to interphase egg extracts supplemented with radiolabeled S-[methyl-3H]-adenosyl-L-methionine and buffer (control), 3×FLAG-mDPPA3, or 3×FLAG-hDPPA3. The efficiency of DNA methylation maintenance was assessed by the incorporation of radio-labeled methyl groups from S-[methyl-3H]-adenosyl-L-methionine (3H-SAM) into DNA purified from the egg extracts. Data were presented as mean values ± SD for n = 3.