Recent studies revealed that a protein known as Hat1p Interacting Factor-1 forms a complex with Hat1p/Hat2p in the nucleus and functions as a histone chaperone during chromatin assembly. This protein is a yeast homolog of the N1/N2 histone chaperone, which functions in both the storage and assembly of histone H3/H4 tetramers during the rapid rounds of DNA replication which occurs early in X. laevis embryogenesis. Hif1p functions as a chromatin assembly factor in vitro and associates with acetylated histone H4 in vivo in a Hat1p/Hat2p dependent manner. These findings demonstrated a physical connection between type B HATs and factors directly involved in the process of chromatin assembly. We have performed biochemical experiments to further characterize this protein. By using chromatographic techniques, we demonstrated that Hif1p forms complexes in both Hat1p/Hat2/-dependent and Hat1p/Hat2p-independent manners. We have developed a method combining both conventional and affinity chromatography to isolate and identify proteins associate with Hif1p. Our results also suggested a link between Hif1p and a H3-specific type B HAT.
The human homolog of Hif1p, NASP, has been reported to be an H1-specific histone chaperone when all the other members of N1/N2 family are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirmed that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicated that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.
We have also further characterized this protein in detail by directly mapping domains that are involved in interactions with each histone in vitro and developing a cell culture model to understand how the association with specific histones contributes to the cellular function of NASP in vivo. We identified two distinct domains that separately interact with linker histone or core histone through different mechanisms. We demonstrated that loss of native NASP increased the sensitivity of chromatin to digestion with micrococcal nuclease, therefore, identified NASP as a significant contributor to global chromatin structure.