The most abundant form of Hat

The most abundant form of Hat1p in the nucleus is apt to be in the form of the NuB4 complex [96]. In the context of PD 0332991 assembly, the function of the NuB4 complex may be similar to that ascribed for the cytoplasmic HAT1 complex. That is, the NuB4 complex may be involved in directing newly synthesized histone H3/H4 complexes to Asf1 for subsequent distribution to replication-coupled and replication-independent chromatin assembly pathways. As discussed above, the majority of the NuB4 complex and Asf1 are nuclear and much of the histone H4 found associated with Asf1 is diacetylated [11], [105]. Another intriguing possibility is that the NuB4 complex may perform functions in the nucleus beyond its role in the metabolism of newly synthesized histones. In addition to histone deposition, Asf1 plays an important role in the eviction of histones from chromatin in the context of transcriptional regulation [119], [123], [124], [125]. It is not known how these histones are recycled into the pathway of chromatin assembly but the association of Asf1 with the NuB4 complex may be an indication that some recycled histone H4 may need to reacquire the deposition-related pattern of acetylation prior to its reassembly into chromatin. While the available evidence suggests that the NuB4 complex functions immediately upstream of Asf1, the importance of this interaction is not known as Hat1 has not been demonstrated to directly influence either replication-coupled or replication-independent chromatin assembly. However, an analysis of the chromatin reassembly that accompanies the recombinational repair of a DNA double strand break has provided a model system to directly examine the role of the NuB4 complex. Using an inducible HO endonuclease that generates a single double strand break at the MAT locus, Tyler and colleagues used chromatin immunoprecipitation (ChIP) to monitor levels of histone H3 near the double strand break. As expected, the levels of H3 decrease as single-strand resection occurs. As double strand break repair is completed, histone H3 levels are restored. Importantly, the restoration of H3 required the presence of Asf1p and histone H3 lysine 56 acetylation confirming the utility of this model for the study of DNA repair-linked chromatin assembly [126]. Loss of HAT1 and HIF1 had previously been shown to cause defects in the recombinational repair of DNA double strand breaks [96], [127]. An involvement in DNA repair is an evolutionarily conserved characteristic of these NuB4 complex components [59], [115], [128]. In addition, a direct involvement of the NuB4 complex in the recombinational repair of DNA double strand breaks was suggested by the fact that both Hat1p and Hif1p are recruited to chromatin near the sites of DNA damage [58]. By monitoring H3 levels at the MAT locus following an HO-induced DNA double strand break, the loss of Hat1p and Hif1p were both shown to result in a significant decrease in the reassembly of chromatin structure following DNA repair, marking the first direct demonstration of a role for the NuB4 complex in a chromatin assembly process. In addition, this system also provided evidence that the NuB4 complex does not exclusively function upstream of Asf1p. Combining deletions of either HAT1 or HIF1 with a deletion of ASF1 (in addition to specific mutations in histone H3) caused an increase in sensitivity to the HO-induced double strand break and an increased defect in DNA repair-linked chromatin reassembly [98]. Therefore, at least in the context of DNA repair, the NuB4 complex may perform functions in chromatin assembly that are independent of Asf1p. One possibility is that Hif1p, which has been shown to function in chromatin assembly assays in vitro, may be part of a distinct chromatin assembly pathway. Alternatively, the NuB4 complex may be capable of shuttling histones into other chromatin assembly pathways in the absence of Asf1p. Our understanding of the function of Hat1 in chromatin assembly has grown dramatically in the past several years. However, many important issues remain to be addressed. Foremost among these is a reconciliation of the biochemical and genetic analyses of Hat1. At the biochemical level, Hat1 and its associated complexes are highly conserved and appear to be among the primary histone H3/H4 binding proteins in most eukaryotic organisms examined [42], [43], [61], [99], [104], [105], [112], [129]. In addition, Hat1 generates an evolutionarily conserved pattern of modification that is found on a very high percentage of newly synthesized histones [5], [11], [105]. Despite this apparent involvement in the histone deposition pathway, Hat1 is not essential for viability in any of the cell types in which it has been genetically deleted (S. cerevisiae, Schizosaccharomyces pombe and chicken DT40 cells) [50], [51], [59], [115]. Therefore, uncovering the complete picture of how Hat1 is integrated in the process of chromatin assembly, which may involve in vivo analyses in a wider variety of organisms, is an important goal. In addition, while it has now been shown that Hat1 is directly involved in the reassembly of chromatin structure that accompanies DNA double strand break repair, it is not known how this reassembly is related to other types of chromatin assembly pathways in the cell. Hence, it will be important to determine whether Hat1, and its associated factors, also participate in replication-coupled and replication-independent chromatin assembly. Finally, studies involving Hat1 have focused on its role in the formation and regulation of chromatin structure. However, it is now apparent that acetylation is a widely used modification that is involved in many cellular processes [36], [130], [131]. Probing the role of Hat1 in non-histone acetylation may uncover novel cellular functions for the original histone acetyltransferase.