Using RITE as a biochemical-genetic pulse-chase tool, we previously observed rapid exchange of histone H3 in chromatin in yeast cells outside S-phase 
. Similar results have been reported using an inducible pGAL-system to overexpress an ectopic tagged histone H3 copy 
. By using RITE, in contrast to the pGAL system, the tagged old and new histone H3 species are expressed by the endogenous H3 promoter from the endogenous chromosomal location 
. Therefore, the high levels of histone exchange observed with RITE were not caused by misregulation of histone H3 expression. Indeed, qRT-PCR and microarray analyses showed that RITE strains containing tagged H3 express very similar H3 mRNA levels as wild-type cells containing untagged H3 at different phases of the cell cycle 
). Interestingly, although histone mRNAs are cell cycle regulated and peak in S-phase when the demand for new histones is highest 
, histone H3 mRNA expression is still relatively high outside S-phase, providing an explanation for the abundant synthesis of new histones outside S-phase 
. To investigate the biological function of histone turnover and its consequences for chromatin structure and function, we developed the Epi-ID barcode screen for chromatin regulators and combined it with RITE. In this screen we found that Hat1 and subsequently also the other members of the NuB4 complex positively regulate histone turnover. To our knowledge, our data provide the first evidence that a Type B histone acetyltransferase complex regulates histone assembly in vivo.
Hat1 was the first histone acetyltransferase identified 
. It is part of a multi-subunit complex that interacts with histone chaperones and acetylates free histones but is inactive towards nucleosomal histones 
. The biological significance of these biochemical activities of the Hat1 complexes remained elusive 
although in genetic tests Hat1 was found to play a role in gene silencing and DNA damage response 
. However, manifestation of these phenotypes required additional mutations in the N-terminal tail of histone H3 and whether these chromatin-related phenotypes are related to histone deposition defects remained unknown.
The known and conserved substrates of HAT-B/NuB4 are lysines 5 and 12 of histone H4 
. Mutation of these residues has revealed functions in histone H4 nuclear import and chromatin assembly 
. However, H4K5,12 mutants generally show no major growth phenotypes or global changes in chromatin organization 
. Here we found a positive effect of H4K5,12A and H4K5,12Q mutants on histone turnover in promoters, suggesting that NuB4 may exert its turnover function via H4K5/K12 acetylation. However, H4K5,12R, mimicking the hypo-acetylated state of these lysines, did not cause a turnover defect (). One possible explanation of these results is that NuB4 has additional substrates that contribute to its role in histone turnover 
. We do not know whether other substrate lysines on histones or perhaps non-histone proteins are also involved and play roles redundant with the acetylated histone H4 tail.
The nuclear function for HAT-B in histone turnover () indicates that HAT-B's role in histone metabolism may be more complex than previously anticipated and extends beyond the acetylation of newly synthesized histones. This is in line with observations that Hat1 can be recruited to chromatin at origins of replication and DNA double strand breaks 
and with the role of members of the NuB4 complex in depositing histones following repair of a DNA double strand break 
. Unexpectedly, our studies revealed that Hat1 and Hat2 act in parallel with Hif1, and that Hat1 and Asf1 bind a different subset of the soluble histone pool. In previous studies Hat1/Hat2, Hif1, and Asf1 have been shown to bind to each other 
, which led to the suggestion that Asf1 acts downstream of Hat1/Hat2/Hif1 and passes on new histones acetylated on H4K5/K12 (and H3K56) to chromatin assembly factors CAF-I, HIR, and Rtt106 
. Our results suggest that Hat1/Hat2, Hif1 and Asf1 act, at least in part, via distinct pathways of chromatin assembly and/or disassembly ( and ). The equal binding of Asf1 to new and old histones suggests that Asf1 may be involved in depositing as well as escorting histones evicted from chromatin (), which is in concordance with the finding that H3K56 acetylation (mediated by Rtt109/Asf1) is a mark of new histones, yet is important for histone eviction and nucleosome destabilization 
. Indeed, histone chaperones may not exclusively function in chromatin assembly 
. For example Nap1, which can escort H3/H4 and H2A/H2B and assemble histone octamers into nucleosomes, but may orchestrate this by promoting nucleosome disassembly 
. Another example is CAF1, which is involved in replication-coupled assembly of new histones into chromatin, yet histone H3 bound to this complex (or to Rtt106 or Asf1) contains methylated H3K79 
, which is a mark of chromatin-bound histones 
What are the functional consequences of altering histone turnover? Histone turnover might affect several aspects of the epigenome, such as nucleosome occupancy, DNA accessibility, or dynamics of histone modifications. No changes in growth or cell cycle progression were observed for single, double, or triple hat1Δ
mutants (Figure S8
) and no significant transcriptional changes were observed (see and Material and Methods
). Apparently, slowing down turnover of histone H3 by loss of the NuB4 complex has no profound consequences under these conditions. Deletion of HAT2
resulted in a moderate increase in expression of the genes encoding histone H3 and H4 in mid-log cultures (). We expect that this may be a response to the histone turnover defects caused by deletion of Hat2 and Hif1, since deletion of Hat1, which overall has a lower impact on histone turnover, did not affect histone gene expression. It is possible that the phenotypes of the hat1Δ
strain are relatively weak because of compensation of Hat1's activity by other HATs, such as Gcn5, which acetylates newly synthesized histone H3 
. In our microarray analyses, we did not observe significant upregulation of mRNA levels of other (putative) HATs in G0 cultures (Figure S9
). Overall, our results indicate that loss of NuB4 function alone has no major consequences for global chromatin organization.
What is the function of histone turnover? Histone turnover leads to turnover of histone modifications and can thereby affect the pattern as well as dynamics of the epigenome. When a chromatin state is controlled by two opposing activities (e.g. modification and demodification by turnover) this could lead to a more rapid establishment a new equilibrium after perturbation of the epigenome, such as during DNA replication or after exposure to stress (e.g. see 
). Based on models proposed for histone acetylation one could also envision that dynamic turnover (cycles of modification and demodification) rather than the steady state may be relevant for chromatin function 
. Alternatively, histone turnover could counteract the accumulation of histone modifications that are less susceptible to demodification. For example, methylation of histone H3K79, which accumulates in a non-processive manner on aging histones 
is enriched in genomic regions that show low histone turnover and retain old histone H3 molecules, suggesting that histone inheritance and dynamics help shape the epigenome 
. The identification of additional mutants in future screens will help to further deconstruct the pathways of histone turnover and to discover their biological significance.
In the Epi-ID screen we also identified Gis1 and Nhp10 as negative regulators of histone turnover. Gis1 is a zinc-finger transcription factor involved in regulation of stress genes 
and contains a Jumonji domain, which has been associated with histone demethylase activity 
. Gis1 has also been reported to bind to several factors involved in DNA metabolism 
. It will be interesting to test whether any of these Gis1-binding proteins or its putative demethylase activity is involved in this novel function of Gis1. Nhp10 is a non-essential subunit of the essential INO80 chromatin remodeling complex that can move or mobilize nucleosomes. Two recent studies suggest a role for INO80 in redeposition of histones during induced transcription 
. That Nhp10 slows down histone turnover provides further support for the idea that the INO80 complex can help to preserve the chromatin architecture during transcription. In an Epi-ID screen using 1536 chromosome biology mutants in which the old and new tags on histone H3 were swapped (old-T7 and new-HA), NHP10
mutants also showed more histone turnover (data not shown), indicating that the phenotypes observed were not caused by tag-specific effects and that Epi-ID can be scaled up.
The application of Epi-ID is not restricted to histone turnover. In fact, in screens for other epigenetic marks such as histone modifications or nucleosome occupancy Epi-ID, can be applied without the elaborate genetic crosses and genetic switches that are required for screens based on the RITE pulse chase assay. Future applications in yeast may benefit from other barcoded mutant collections that are being developed 
. Although our study suggests that position effects of the barcoded marker are not major confounders in Epi-ID and can be (at least in part) excluded by comparing UpTag with DownTag barcodes, DNA barcodes at a common genetic locus separated from the gene deletion would be preferable for epigenetic screens. The recently developed Yeast Barcoders Library represents such a collection in which barcoded markers are integrated at the common HO locus thereby providing opportunities to further expand and improve the application of Epi-ID in yeast 
. Finally, the basic principles of this approach should also be applicable to barcoded mutant libraries in other organisms, such as barcoded episomes, or transposon or virus insertion libraries.