Tian W et al. (2019), The HRP3 PWWP domain recognizes the minor groov...

XB-ART-56530

Nucleic Acids Res 2019 Jun 04;4710:5436-5448. doi: 10.1093/nar/gkz294.

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The HRP3 PWWP domain recognizes the minor groove of double-stranded DNA and recruits HRP3 to chromatin.

Tian W , Yan P , Xu N , Chakravorty A , Liefke R , Xi Q , Wang Z .

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HDGF-related protein 3 (HRP3, also known as HDGFL3) belongs to the family of HDGF-related proteins (HRPs) and plays an essential role in hepatocellular carcinoma pathogenesis. All HRPs have a PWWP domain at the N-terminus that binds both histone and DNA substrates. Despite previous advances in PWWP domains, the molecular basis by which HRP3 interacts with chromatin is unclear. In this study, we solved the crystal structures of the HRP3 PWWP domain in complex with various double-stranded DNAs with/without bound histone peptides. We found that HRP3 PWWP bound to the phosphate backbone of the DNA minor groove and showed a preference for DNA molecules bearing a narrow minor groove width. In addition, HRP3 PWWP preferentially bound to histone peptides bearing the H3K36me3/2 modification. HRP3 PWWP uses two adjacent surfaces to bind both DNA and histone substrates simultaneously, enabling us to generate a model illustrating the recruitment of PWWP to H3K36me3-containing nucleosomes. Cell-based analysis indicated that both DNA and histone binding by the HRP3 PWWP domain is important for HRP3 recruitment to chromatin in vivo. Our work establishes that HRP3 PWWP is a new family of minor groove-specific DNA-binding proteins, which improves our understanding of HRP3 and other PWWP domain-containing proteins.

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Species referenced: Xenopus laevis
Genes referenced: aopep ctbp1 hdgf

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	Figure 1. Apo-form structure of the HRP3 PWWP domain and its DNA binding analysis. (A) Domain architecture of the HRP family of proteins. (B) Apo-form structure of the HRP3 PWWP domain. The PWWP domain is coloured green. Residues forming the aromatic cage are coloured pink. The MES molecule is coloured yellow. (C) Electrostatic surface view of HRP3 PWWP. The positively charged surface is coloured blue. The negatively charged surface is coloured red. (D) EMSA analysis of the binding of HRP3 PWWP to various dsDNAs. The protein to DNA molar ratio is listed above the lanes. (E) MST-based measurements of the binding affinities of HRP3 PWWP to various dsDNAs. Dissociation constants (Kd) are listed within the panel.
	Figure 2. Structural details of the HRP3 PWWP and dsDNA complex and related mutational analysis. (A) Structure of the HRP3 PWWP and 16-mer-TA dsDNA complex. (B) Schematic representation of the interactions between HRP3 PWWP and the 16-mer-TA DNA. The DNA sequence is arbitrarily assigned. (C) Details of the interaction between HRP3 PWWP and the minor groove of the 16-mer-TA DNA. (D) Ribbon representation of the overlapped structures of the PWWP/TA-rich-DNA and PWWP/GC-rich-DNA complexes. In the TA-rich complex, PWWP is coloured green and the bound DNA is coloured orange. In the GC-rich complex, PWWP is coloured blue and the bound DNA is coloured grey. (E) Graphs comparing the minor groove widths (Å) and electrostatic potentials (kT/e) for 16-mer-TA and 10-mer-GC DNA molecules. (F) MST-based measurements of the dissociation constants of wild-type or mutant HRP3 PWWP for 16-mer-TA DNA (left panel) or 16-mer-GC DNA (right panel). (G) A table listing the dissociation constants measured in (F).
	Figure 3. HRP3 PWWP binds to the H3K36me3/2-containing histone peptide. (A) A panel of overlapping HSQC spectra of the HRP3 PWWP domain with various concentrations of the H3K36me3 peptide. (B) A table listing the NMR-based measurements of the dissociation constants for the binding between the wild-type or mutant forms of HRP3 PWWP and various H3K36-containing peptides with/without modifications. (C) NMR-based measurements of different dissociation constants of HRP3 PWWP interacting with H3K36me3/2/1/0-containing histone peptides. (D) Structural details of the ternary complex of HRP3 PWWP/dsDNA/H3K36me3-peptide. The histone peptide is coloured yellow. (E) Structural details of the ternary complex of HRP3 PWWP/dsDNA/H3K36me2-peptide. (F) NMR-based measurements of the dissociation constants for the binding of the different HRP3 PWWP mutants with the H3K36me3 peptides. (G) Overlapped structures of the PWWP domain from HRP3 (in green), Brpf1 (in cyan) and DNMT3B (in magenta) with bound H3K36me3 peptides.
	Figure 4. Model of HRP3 PWWP recruitment on the H3K36me3-containing nucleosome. (A) EMSA analysis of HRP3 PWWP with the unmodified nucleosome or with the H3KC36me3-modified nucleosome. +1 or +2 indicates one or two HRP3 PWWP binding to a single nucleosome. (B) A side overview of the HRP3-nucleosome model. The PWWP domain is coloured green. PWWP-bound DNA coloured in grey is docked into a region of the SHL1 minor groove. Histone H3 N-terminal tails are coloured salmon. (C) A zoomed view of the HRP3-nucleosome model. The N-terminal H3 tail, HRP3 PWWP and its bound DNA are coloured the same as in (B). HRP3-bound histone peptide is coloured cyan. The residues H3K36 and H3R40 in the nucleosome and H3K36me3 in the HRP3 complex are shown as spheres.
	Figure 5. HRP3 binds to accessible chromatin. (A) Fluorescence microscopy of GFP-tagged HRP3 proteins in HepG2 cells, in comparison to GFP control. Scale bar, 100 μm. (B) Distribution of various ectopically expressed GFP-HRP3 variants in nucleoplasmic or chromatin fractions of HepG2 cells. (C) Genome-wide HRP3 distribution compared to the genome. (D) Three gene groups were identified based on HRP3 levels. Group I genes are characterized by very strong binding up- and downstream of the transcription start site. Group II genes have HRP3 mainly bound at the promoter region. Group III genes are not bound by HRP3. (E) Profiles of HRP3 and H3K36me3 in the three gene groups. (F) Relationship between HRP3 and H3K36me3 at gene bodies. (G) Heat maps showing all significant HRP3 peaks (n = 79 555), sorted by size, and the respective DNase I hypersensitivity and nucleosome density. (H) HRP3 levels of the wild-type and mutant HRP3 (KNR-A = K18A/N76A/R78A, KYNR-A = K18A/Y22A/N76A/R78A) at gene bodies of group I genes and promoters of group II genes. (I) Example at the CTBP1 gene.

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