Systematic Study On Binding Properties Of Human PWWP Domain To Histone And DNA

Background:Chromatin plays an essential role in the regulation of gene transcription,cell cycle progression,DNA replication and repair processes.In eukaryotes,nucleosomes are the basic units of chromatin,formed by DNA duplexes wrapped around octamers of histones.The N-terminal of histones are susceptible to a variety of post-translational modifications,and lysine methylation,one of the major post-translational modifications,plays an important role in regulating chromatin structure and controlling gene expression.Many proteins have been reported to recognize post-translational modifications of histones,such as histone methylation by members of the Royal family and histone acetylation by bromodomain.The PWWP domain(with about 100-150 amino acid residues),a member of the Royal family,has a typical aromatic cage that recognizes methylated histones,and can also bind to double-stranded DNA(dsDNA).The PWWP domain-containing proteins bind to nucleosomes by binding to methylated histones and dsDNA synergistically,and localize to chromatin,where they function in biological processes such as DNA replication,transcription,repair,and pre-mRNA processing.The NSD family proteins,includeing NSD1,NSD2,and NSD3,are lysine methyltransferases for monomethylation and dimethylation of histone H3K36.They all contain two PWWP domains(N-terminal and C-terminal PWWP,or PWWP1 and PWWP2,respectively),and the N-terminal PWWP domains of NSD2 and NSD3 have been proved to bind to H3K36me3.Several studies have revealed that NSD methyltransferases are overexpressed,amplified or somatically mutated in several types of cancers,suggesting that NSD methyltransferases play an important role in the occurrence and development of cancer.Aim:Systematic investigation of the binding abilities of human PWWP domains to dsDNA and histones.Results:In this study,we first used the representative human PWWP domain constructed by our research group for protein expression and purification,and systematically analyzed the binding abilities of human PWWP domains to dsDNA by protein binding microarray(PBM)experiments and isothermal titration calorimetry(ITC).Our results showed that all purified PWWP domains are able to bind to dsDNA in a sequence-independent manner and the binding affinity is dependent on dsDNA length,the longer the dsDNA,the higher the affinity.In addition,we examined the binding ability of the N-terminal and C-terminal PWWP domains of NSD subfamily proteins to trimethylated histone peptides at different sites by fluorescence polarization assay(FP),and found that both PWWP domains of NSD2/3 preferentially recognize H3K36me3 and H3K79me3.Our ITC data showed that both PWWP domains of NSD2 and NSD3 are able to bind to dsDNA without sequence selectivity too.In order to investigate the molecular mechanism of PWWP domain interaction with dsDNA and histones,we used the purified PWWP domain protein to co-crystallize with dsDNA and histone peptides,and finally obtained the structures of BRPF2_PWWP in complex with dsDNA,NSD3_PWWP2 in free state and NSD2_PWWP1 in complex with dsDNA by X-ray diffraction and crystal structure determination.The crystal structure of the BRPF2_PWWP domain in complex with 12 mer-dsDNA showed that the PWWP domain interacts with dsDNA by binding to its major groove,instead of the minor groove observed in the HDGF family of PWWP domains.Our study indicates that PWWP domains could bind to dsDNA in different modes,namely,binding to the phosphate backbone of the major or minor groove of dsDNA in a sequence independent manner.NDS3_PWWP2 and NSD2 PWWP1 domains crystal structures indicate that they both contain a conserved N-terminalβ-barrel formed by five β-strands,a long C-terminal α-helix for NSD3 and a C-terminal α-helix bundle consisting of four α-helices for NSD2,respectively.In addition,an extra α-helix is inserted between β2 and β3 of NSD3_PWWP2,and an extra β-fold is inserted into the same position of NSD2_PWWP1.Futher structural analysis showed that they both have a significant positive charged surface for dsDNA binding and a conserved aromatic cage for methylated histones binding.Structural superposition with the PSIP1-nucleosome complex revealed that they may require conformational adjustment to interact with nucleosome.Conclusion and significance:PWWP domains bind to dsDNA without sequence selectivity,and the binding affinity depends on dsDNA length,the longer the length,the stronger the affinity.The crystal structure of the BRPF2 PWWP domain in complex with dsDNA reveals that the PWWP domain interacts with dsDNA by binding to its major groove,instead of the minor groove observed in the HDGF family of PWWP domains.Our study indicates that PWWP domains could bind to dsDNA in different modes.In addition,our studies also indicate that all the PWWP domains of NSD proteins except NSD1_PWWP1 bind to trimethylated H3K36,H3K79 peptides and dsDNA weakly.Our crystal structures uncover that the NDS3_PWWP2 and NSD2_PWWP1 domains,which hold an extremely long α-helix and α-helix bundle,respectively,need a conformation adjustment to interact with nucleosome.Our studies lay a foundation for further research on how PWWP domain-containig proteins synergistically bind to dsDNA and histone to target nucleosomes.