Maintaining the dynamic structure of chromatin is critical for regulating the cellular processes that require access to the DNA template, such as DNA damage repair, transcription, and replication. Histone chaperones and ATP-dependent chromatin remodeling factors facilitate transitions in chromatin structure by assembling and positioning nucleosomes through a variety of enzymatic activities. SMARCAD1 is a unique chromatin remodeler that combines the ATP-dependent ability to exchange histones, with the chaperone-like activity of nucleosome deposition. We have shown previously that phosphorylated SMARCAD1 exhibits reduced binding to nucleosomes. However, it is unknown how phosphorylation affects SMARCAD1's ability to perform its various enzymatic activities. Here we use mutational analysis, activity assays, and mass spectrometry, to probe SMARCAD1 regulation and to investigate the role of its flexible N-terminal region. We show that phosphorylation affects SMARCAD1 binding to nucleosomes, DNA, and histones H2A-H2B as well as ATP hydrolysis and histone exchange. Conversely, we report only a marginal effect of phosphorylation for histone H3-H4 binding and nucleosome assembly. In addition, the SMARCAD1 N-terminal region is revealed to be critical for nucleosome assembly and histone exchange. Together, this work examines the intricacies of how phosphorylation governs SMARCAD1 activity and provides insight into its complex regulation and diverse activities.
