Identification of distinct SET/TAF-1β domains required for core histone binding and quantitative characterisation of the interaction

Background: The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-1 beta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-1 beta, we designed several SET/TAF-1 beta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. Results: Wild type SET/TAF-1 beta binds to histones H2B and H3 with K-d values of 2.87 and 0.15 mu M, respectively. The preferential binding of SET/TAF-1 beta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-1 beta, as well as the H3 amino-terminal tail, are dispensable for this interaction. Conclusion: This type of analysis allowed us to assess the relative affinities of SET/TAF-1 beta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-1 beta and can be valuable to understand the role of SET/TAF-1 beta in chromatin function.