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  • The NuB complex interacts with Asf p

    2021-10-14

    The NuB4 complex interacts with Asf1p, which plays a central role in controlling the flux of histones to the various chromatin assembly pathways. This interaction is also evolutionarily conserved having been detected in yeast, chicken and human I-BET151 [27], [61], [104], [105]. Asf1p appears to interact with the NuB4 complex at substoichiometric levels. When Asf1p is isolated by affinity purification, the presence of NuB4 complex components can be detected by Western blot analysis but are difficult to observe by either coomassie blue or silver staining [27]. Likewise, Asf1p is not readily apparent in purified Hat1p complexes [50], [65], [96], [106]. The interaction of Asf1p with the NuB4 complex requires the presence of both of its histone chaperones (Hat2p and Hif1p) and appears to be mediated by their interactions with histones [27], [61]. These histones are likely to interact with the NuB4 complex prior to their association with Asf1p based on the observation that the bulk of the histone H4 that is bound to Asf1p is acetylated on lysines 5 and 12 [61], [105]. It is not clear where the interaction of the NuB4 complex with Asf1p occurs. As is the case for the NuB4 complex itself, the NuB4–Asf1p complex has been detected in human cytosolic extracts [61], [105], [107]. Again, as these factors are predominantly nuclear, the presence of these complexes in cytoplasmic extracts may represent a transient intermediate in the histone deposition process or result from an artifact of the fractionation process. The Hat1 core complex also associates with nuclear factors independent of the NuB4 complex. Using a TAP-tagged Hat1p, Suter and colleagues, used affinity purification to identify proteins that physically interact with Hat1p. In addition to the expected Hat2p and Hif1p, all six subunits of the origin recognition complex (ORC) co-purified with Hat1p. Reciprocal affinity purification of the ORC complex then showed that only the Hat1 core complex (Hat1p and Hat2p) was actually associated with ORC. As was the case with Asf1p, ORC was present in levels far below those of Hat2p and Hif1p. A number of observations suggest that the physical interaction between the Hat1 core complex and ORC is functionally relevant. First, deletion of the HAT1 or HAT2 genes produced synthetic growth and viability defects when combined with temperature sensitive alleles of ORC2 and ORC5. Second, although the interaction between ORC and the Hat1 core complex did not vary throughout the cell cycle, Hat1p displayed an S-phase specific localization to chromatin at origins of replication. However, I-BET151 the role of the Hat1 core complex at origins is not known as the absence of Hat1p did not lead to any alterations in DNA replication [108]. Perhaps Hat1p acts to modify histone acetylation levels at origins (analogous to the role of Hbo1 in mammalian cells) or that the interaction with ORC may help to localize the Hat1 core complex to sites of DNA replication to participate in replication coupled chromatin assembly but that functional redundancy exists with other HATs or with the acetylation of histone H3 masking the effect of Hat1p loss [109], [110].
    Histone chaperone activities of HAT1-containing complexes Perhaps the most surprising components of the Hat1-containing complexes are histone H4 and histone H3. Enzymes are typically involved in transient interactions with their substrate. The enzyme will bind to the substrate, perform the relevant catalysis and then release the reaction product. However, Hat1 does not appear to function in a strictly catalytic fashion and appears to act in a somewhat stoichiometric manner by serving as a major histone interacting protein. This was first observed in experiments in which an epitope tagged histone H4 was affinity purified from yeast cytoplasmic extracts. The primary proteins associated with the cytoplasmic histone H4 were Hat1p, Hat2p, histone H3 and Kap123p (a karyopherin/importin) [111]. Histone H3 and H4 were also found to be associated with the NuB4 complex isolated from yeast nuclei [96]. Hat1 has also been found to co-purify with the soluble fraction of epitope tagged histones H3 and H4 from a variety of other organisms that include P. polycephalum, chickens and humans [42], [61], [99], [104], [112], [113]. These soluble histone fractions have been isolated from both cytosolic and nuclear extracts and the other components of the NuB4 complex (Hat2/Rbap48 and Hif1p/NASP) are often found associated with the histones as well. Hence, a picture has emerged in which Hat1 not only associates with histone H4 soon after its synthesis but also remains bound to the histone throughout much of the deposition pathway.