Structure And Function Of The PHD5-C5HCH Module Of Human NSD Family

The NSD family of SET domain-containing histone methyl-transferases includes NSD1, NSD2(also known as WHSC1, Wolf-Hirschhorn syndrome candidate1or MMSET, multiple myeloma SET), and NSD3(also known as WHSC1L1, WHSC1like1). All three NSD proteins have been directly linked to multiple human diseases. Although highly relevant to human health and diseases, only a few details have been published regarding the mechanism of NSD family action.A striking feature of the three NSD proteins is that they are highly similar within the block of about700amino acids, which contains a catalytic SET domain with its pre-and post-domains, a PWWP domain, five PHD fingers, and an NSD-specific Cys-His rich domain (C5HCH). However, the similar domain architecture of the three NSD family members does not indicate a functional redundancy. The SET domains of the three NSD proteins have been confirmed to have highly specific H3K36mono-or dimethylase activities. H3K36methylation has been implicated in multiple biological processes, and the biological consequences of such modification are largely determined by the location of the gene where the methylated H3K36mark is placed. Because the NSD proteins also contain several histone Readers, one attractive possibility is that these Readers recruit the NSD H3K36methyltransferases to their specific gene sites, thus lead to distinct biological outcomes. Therefore, the molecular mechanishm of the Reader domains to recognize the chromatin is critical to undertand their biological impact and the rationale of functional redundancy of NSD proteins.Herein, we used several in vitro binding assays to identify the histone binding ability of the C-terminal PHD5-C5HCH module of NSD3. We demonstrate that the PHD5-C5HCH module of NSD3prefers to bind H3N-termianl peptide containing unmodified H3K4and trimethylated H3K9. We solved the high resolution crystal structures of PHD5-C5HCHNSD3in its apo and holo states with unmodified H3residues1-7(H31-7),1-15(H31-15), and1-15with lysine9trimethylated (H31-15K9me3), respectively. These structures reveal an integrated tandem PHD-PHD-like fold with H3peptide bound only on the surface of PHD5and provide the structural basis for the peptide recognition by PHD5of NSD3. Further mutagenesis and peptide binding assays show that PHD5-C5HCHNSD1does not bind to H3, and the PHD5-C5HCH module of NSD2(PHD5-C5HCHNSD2) prefers to bind to unmodified H3K4and H3K9. This is likely due to a minor sequence change at the H3binding surface. Our findings suggest that PHD5-C5HCHs of the NSD family are conserved in the overall structure but vary in H3recognition. These variable recognitions may play a role in the localization of these H3K36methyltransferases to different genome sites and are consistent with the distinct and non-redundant functions of NSD proteins. Our results also imply a link between the deposition of H3K36methylation and the recognition of H3K4and H3K9.