In this study we systematically surveyed the functional associations among genes that dynamically regulate histone acetylation and deacetylation in yeast, generating a comprehensive network of genetic interactions. The inclusion of six essential query genes significantly enhanced our ability to identify functionally important target genes.
Our analysis focused on the abstracted network of functional modules and protein complexes rather than those of individual genes. The network was highly connected and revealed a close functional relationship between HAT and HDAC complexes, indicating that these complexes share common essential cellular functions despite the fact that most of them modify distinct spectra of histone lysine residues. The HDA complex was the most distinct SR hub, pointing to major counter-balancing effects on the NuA4, SAGA and Elongator HAT complex, which indicates that the HDA complex removed the largest amount of acetyl groups. By contrast, the Rpd3C had aggravating interactions with most of the HAT complexes, except Piccolo NuA4; these interactions are consistent with the cooperation between Rpd3C(S) and various HATs in regulating transcriptional elongation (Carrozza et al. 2005
; Keogh et al. 2005
; Li et al. 2007
), and also the role of Rpd3C in governing histone H4 deacetylation following an acetylation conducted by NuA4 in the vicinity of DNA DSBs.
Our results also revealed the general finding that the HDA and Rpd3 complexes together define the major histone deacetylase activities that counteract the histone acetyltransferase activity of the NuA4 and SAGA complexes, and that these complexes provide the bulk control of the dynamic balance of global histone acetylation and deacetylation essential to cell viability. Requirement of histone acetylation by various HATs for maintaining cell viability have been well studied (Smith et al. 1998
; Zhang et al. 1998
; Allard et al. 1999
; Clarke et al. 1999
; Howe et al. 2001
). Our data suggest that hyperacetylation of histone H3 and H4 in the hda1Δ rpd3Δ
double mutant is as detrimental to cell viability as hypoacetylation, and can be rescued when the responsible acetylase activity is repressed.
In addition to its effect on many aspects of chromosome biology, a relationship between global histone acetylation/deacetylation and vacuolar function is also revealed by these studies. This relationship raises the possible existence of nonhistone substrates of HATs and HDACs in yeast.
The genetic interaction profile of htz1Δ
and follow-up experiments led us to conclude that the HDA complex, previously known to acetylate histones H3 and H2B, removes the acetyl group from K14 and possibly also other lysine residues of the N-tail of Htz1p. However, lack of reliable antibodies limited our ability to test the acetylation level of lysine residues other than K14. We also propose that histone H3 and Htz1p reside in the same functional module based on their similar genetic interaction profiles. However, although being less enriched in the promoter region and defective in blocking telomeric heterochromatin spreading, an unacetylatable Htz1p mutant (htz1-K3,8,10,14R
) is insensitive to genotoxic agents lethal to htz1Δ
, suggesting that acetylation is important in some but not all aspects of Htz1p function (Babiarz et al. 2006
; Millar et al. 2006
). The genetic interactions between HTZ1
and its corresponding HATs (ESA1
) and HDAC (HDA1
) suggest that these complexes not only affect Htz1p modification but also modulate the essential pathway through additional unknown mechanisms.
In addition to examining functional relationships among protein complexes, synthetic genetic interaction profiles can be used for dissecting more elaborate protein complexes like NuA4 into separate functional modules. A recent genetic interaction survey of a subset of nonessential NuA4 subunits revealed that Eaf1p is important for maintaining the integrity of NuA4 complex (Mitchell et al. 2008
). Here, comprehensive incorporation of most essential and nonessential NuA4 subunit genes led to many new findings. The genetic interaction profile of esa1-531
revealed new functions of Esa1p beyond its well-known nucleosomal acetylation activity. For example, we found that the enzymatic activity of Piccolo NuA4, the core acetylation machinery of NuA4, was maintained by control of Esa1p on the protein turnover of Yng2p through an acetylation-dependent mechanism. Deacetylation of Yng2p was dependent on Rpd3p, which potentially precedes the degradation of Yng2p by proteasome. Tandem mass spectrometry and further biochemical experiments confirmed that K170 is the major acetylated lysine residue of Yng2p. The hypersensitivity of both yng2-K170R
mutants (mutations mimicking constitutive deacetylation and acetylation, respectively) to benomyl and MMS suggests that dynamic acetylation and deacetylation is important to its normal function in DSB repair. Moreover, the recruitment kinetics of the Piccolo NuA4 subunits to an HO-induced DSB is distinct from the rest of NuA4. NuA4 is actively recruited focally and rapidly hyperacetylates nearby histone H4 at a DSB. We propose that the dynamic protein turnover of Yng2p mediated by an acetylation-deacetylation cycle with cooperative recruitment of Rpd3C and proteasome at DSBs disrupts the enzymatic activity of Piccolo NuA4, which stops ongoing acetylation. This finding is consistent with previous findings showing that proteasome is involved in the repair of DSBs (Krogan et al. 2004b
). The recruited Rpd3C further removes acetyl groups from histone H4, and ATP-dependent chromatin remodeling complexes actively conduct nucleosome displacement (Tsukuda et al. 2005
). These three mechanisms allow for dynamic hyperacetylation and subsequent hypoacetylation of histone H4 nearby facilitating DSB repair ().
In this paper we have applied genetic interaction analysis on a large scale as a general approach for analyzing the complexities of histone (de)acetylation in yeast. New functions of the NuA4 and HDA complexes were identified, and a potential nonhistone substrate of Esa1p and Rpd3p was found. Extensions of this powerful strategy to mammalian systems will certainly be of interest in light of recent advances in high-throughput methodologies based on RNA interference (Silva et al. 2008