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The NSD proteins, namely NSD1, NSD2 and NSD3, are lysine methyltransferases, which catalyze mono- and di-methylation of histone H3K36. They are multi-domain proteins, including two PWWP domains (PWWP1 and PWWP2) separated by some other domains. These proteins act as potent oncoproteins and are implicated in various cancers. However the biological functions of these PWWP domains are still largely unknown. To better understand the functions of these proteins' PWWP domains, we cloned, expressed and purified all the PWWP domains of these NSD proteins to characterize their interactions with methylated histone peptides and dsDNA by quantitative binding assays and crystallographic analysis. Our studies indicate that all these PWWP domains 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. • NSD PWWP domains except NSD1_PWWP1 bind to histone H3K36/79me3 and dsDNA weakly. • The NSD3_PWWP2 holds a unique long C-terminal α-helix following the β-barrel. • The NSD2_PWWP1 bears a C-terminal α-helix bundle containing 4 α-helices. • NSD PWWPs interact with methylated histone similar to other reported PWWP domains. • NSD3_PWWP2 and NSD2_PWWP1 need conformational changes to interact with nucleosome. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA binding abilities of Arabidopsis thaliana MBD proteins have been characterized systematically. • AtMBD6 and AtMBD7 serve as the methylated DNA "readers". • Crystal structures of AtMBD6-mCG and apo-state AtMBD5 MBD were determined. • The arginine fingers coupled with the loop1 and α1 of MBD contribute to DNA binding. • This study provides general guidelines in understanding the DNA binding by MBD. 13 MBD-containing genes (AtMBD1-13) have been identified in Arabidopsis thaliana so far, however, their DNA binding ability is still controversial. Here, we systematically measured the DNA binding affinities of these MBDs by ITC and EMSA binding assays, except for those of pseudogenes AtMBD3 and AtMBD13, and found that only AtMBD6 and AtMBD7 function as methylated DNA readers. We also found that the MBD of AtMBD5 exhibits very weak binding to methylated DNA compared to that of AtMBD6. To further investigate the structural basis of AtMBDs in binding to methylated DNA, we determined the complex structure of the AtMBD6 MBD with a 12mer mCG DNA and the apo structure of the AtMBD5 MBD. Structural analysis coupled with mutagenesis studies indicated that, in addition to the conserved arginine fingers contributing to the DNA binding specificity, the residues located in the loop1 and α1 are also essential for the methylated DNA binding of these MBDs in Arabidopsis thaliana , which explains why AtMBD5 MBD and the other AtMBDs display very weak or no binding to methylated DNA. Thus, our study here systematically demonstrates the DNA binding ability of the MBDs in Arabidopsis thaliana , which also provides a general guideline in understanding the DNA binding ability of the MBDs in other plants as a whole. [ABSTRACT FROM AUTHOR]