Abstract:
Chromatin regulation arises from a complex interplay between nucleosome architecture, protein-protein interactions, and precise histone modifications. Nucleosome organization, histone variants such as H2A.Z and mutations in histone H3, modulate DNA accessibility and gene expression. Even subtle alterations in nucleosome structure can profoundly impact the recruitment and function of chromatin-modifying enzymes. Central to these processes are the NSD methyltransferases (NSD1, NSD2, and NSD3), which catalyze methylation of H3K36, a modification essential for transcriptional regulation, development, and oncogenesis.

In this study, we employed high-resolution NMR spectroscopy, biochemical assays, thermal stability measurements, and computational modeling to investigate chromatin dynamics. Replacement of canonical H2A with H2A.Z increased histone tail mobility and reduced nucleosome melting temperatures, while leaving H3-H4 tetramer stability unaffected. These structural changes altered methylation kinetics, with NSD1 displaying a preference for monomethylation and a slower transition to dimethylation on H2A.Z nucleosomes. Binding studies indicated that differences in the acidic patch between H2A and H2A.Z have minimal effects on NSD3 interaction, suggesting that the enhanced tail dynamics of H2A.Z primarily drive enzyme binding.

Structural plasticity among NSD methyltransferases further highlights the complexity of chromatin regulation. While the catalytic cores remain largely rigid, regulatory loops differ significantly: NSD2 exhibits stable loops, whereas NSD1 and NSD3 possess more dynamic loops that influence substrate recognition and catalytic efficiency. Notably, the hyperactive NSD2 E1099K mutant demonstrates pronounced slow, segmental motions and reduced thermal stability, despite maintaining a similar static structure. Druggability assessments of novel inhibitors using NMR-based competition assays confirmed a SAM-competitive mechanism for NSD2, accompanied by structural rearrangements in key regulatory regions.

Oncogenic mutations in histone H3.3, such as G34R/W and K36M, further complicate chromatin regulation. G34 mutations differentially perturb local interactions affecting NSD recognition, whereas K36M abolishes methylation altogether.

Our integrated approach provides new insights into how nucleosome composition influences chromatin dynamics and NSD methyltransferase activity, advancing our understanding of epigenetic regulation and informing the development of targeted therapies for cancer and other diseases.