The factors that modify chromatin structure play important roles in regulating transcription, DNA replication, and repair of DNA damage. In this work we have identified important functional interactions between the FACT chromatin reorganizing factor, the NuA4 histone acetyltransferase, and the two Rpd3 histone deacetylases, Rpd3(L) and Rpd3(S). Our genetic studies show that FACT and NuA4 mutually reinforce one another and that FACT activity is opposed by Rpd3(S) (Fig. ). NuA4 and Rpd3(S) each has at least two subunits involved in binding nucleosomes, and the nucleosome binding subunits of Rpd3(S), Eaf3 and Rco1, recognize methylated K36 of histone H3. Our genetic experiments led to a model where NuA4 and Rpd3(S) compete for binding to nucleosomes (Fig. ), with one consequence of the outcome of the competition being the level of FACT activity required for growth. The model is supported by ChIP experiments showing that mutations eliminating Rpd3(S) result in increased NuA4 binding in vivo and by reciprocal binding of NuA4 and Rpd3(S) at an inducible promoter.
FIG. 10. Model of competition between Rpd3(S) and NuA4. Rpd3(S) and NuA4 each have at least two subunits that mediate association with nucleosomes. Rco1 and Eaf3 each recognize methylated K36 of histone H3. Arp4 is thought to recognize histones in several ways, (more ...)
Our results are consistent with those in previous reports showing that Rpd3(L) and Rpd3(S) have different roles (11
), but it is a surprise to find that they can have opposing functions. Conditions that decrease the ratio of Rpd3(S) to Rpd3(L), such as elimination of the Rpd3(S)-specific subunit Rco1, suppress defects in either FACT or NuA4, and mutations that increase this ratio, such as deletion of SDS3
or overexpression of Rco1, enhance defects in FACT or NuA4. The appropriate distribution of common subunits between Rpd3(S) and Rpd3(L) complexes is therefore important at least partly because these complexes have opposing effects on FACT and NuA4 and therefore the ratio of the two HDAC complexes regulates the activity of these two essential factors.
The need to balance Rpd3(L) and Rpd3(S) is also evident in gcn5
mutants. Here, mutations that favor either Rpd3(L) or Rpd3(S) complex formation are detrimental. Only balanced levels or, more surprisingly, the absence of any Rpd3 complexes is compatible with robust growth. The growth defects of a gcn5
mutant lacking Rpd3(L) can be suppressed by disruption of the SET2
gene encoding a KMT that modifies H3(K36). Rpd3(S) binds to methylated H3(K36) (12
), and preventing this modification and thus making a change in where Rpd3(S) acts is sufficient to overcome the problem caused by the absence of Rpd3(L) in the gcn5
NuA4 and Rpd3(S) bind nucleosomes differently despite the presence in both complexes of the Eaf3 subunit, which binds methylated H3(K36). NuA4 has not been shown to recognize methylated H3(K36) nucleosomes in vitro, while Rpd3(S) does bind such modified nucleosomes in vitro because of the combined action of the chromodomain of Eaf3 and the PHD domain of Rco1 (32
). Thus, Rpd3(S) contains two subunits, Rco1 and Eaf3, that bind to modified histone residues (Fig. ). Rco1 contains a PHD domain that binds methyl
), and the Rco1 PHD domain is required for Rco1 activity in vivo. An rco1
gene disruption suppresses growth defects caused by a FACT mutation, as do rco1
mutants either lacking the PHD domain or with the native PHD domain replaced by a Yng2 PHD domain. Additionally, while overexpression of Rco1 is toxic in certain mutants, overexpression of Rco1(ΔPHD) or Rco1(Yng2-PHD) does not inhibit growth. An EAF3
gene disruption also suppresses the growth defects of FACT mutants. Importantly, NuA4 lacking the Eaf3 subunit displays altered histone acetyltransferase activity in vitro (J. Cote, personal communication). Eaf3 is present in both Rpd3(S) and NuA4, raising the question of how the eaf3
mutation suppresses. However, point mutations in two subunits of NuA4, Esa1 and Arp4, show synthetic defects when combined with FACT mutations, suggesting that the suppressive effect of the eaf3
mutation on FACT mutants is due to the absence of Eaf3 from Rpd3(S).
In addition to Eaf3, NuA4 contains other subunits that may bind histones, including Eaf1 and Eaf2, with SANT domains: Esa1 with a chromodomain and Yng2 with a PHD domain (17
). Whereas the two nucleosome binding subunits in Rpd3(S) are dependent on methylation of histone H3(K36), nucleosome binding by NuA4 is largely independent of K36 methylation (Fig. ). The Set2 enzyme methylates H3(K36), and a set2
mutation eliminates binding of Rpd3(S) to nucleosomes (12
). Importantly, a set2
mutation does not significantly affect binding of NuA4 to nucleosomes in vivo (Fig. and ), presumably due to NuA4 subunits other than Eaf3 that promote association with nucleosomes. While a set2
mutation robustly suppresses both FACT mutants (4
) and FACT NuA4 double mutants (see Fig. S4 at http://www.path.utah.edu/research/labs/david-stillman-lab/supplement
shows either weak suppression or synthetic defects when combined with mutations in the ARP4
(Fig. ) or ESA1
(see Fig. S4 at http://www.path.utah.edu/research/labs/david-stillman-lab/supplement
) subunit of NuA4. Thus, FACT and NuA4 are differently affected by a set2
mutation. The suppression of FACT mutants by a set2
mutation could happen because the absence of methylated H3(K36) prevents Rpd3(S) binding. This idea is consistent with the lack of additivity in suppression by the combination of set2
mutations. Finally, although rco1
mutations are both robust suppressors of a variety of FACT defects, including those of FACT NuA4 double mutants, rco1
both show mild synthetic defects in combination with esa1
mutations (see Fig. S5B at http://www.path.utah.edu/research/labs/david-stillman-lab/supplement
), and thus, rco1
do not suppress all defects in these pathways.
Although our work shows effects of mutations affecting Rpd3(S), Set2, and FACT on transcriptional initiation, previous work has provided functions for these factors in transcriptional elongation. In contrast, it has been shown that Sds3 and Htz1 both localize primarily to promoter elements (27
), and it is possible that sds3
mutations show synthetic defects when combined with FACT mutants because of a linkage between transcriptional initiation and elongation. Further work will be needed to understand whether a defect in transcriptional initiation could affect elongation.
Based on our findings, we developed a model of competition between NuA4 and Rpd3(S) (Fig. ). NuA4 and Rpd3(S) act in opposition, and both complexes contain a common Me-K36 binding subunit, Eaf3. The similar genetic effects of mutating the Set2 methyltransferase or the Rco1 subunit of Rpd3(S) on yFACT and NuA4 mutants suggested there may be competition for binding to methylated H3(K36). To address the question of competition between NuA4 and Rpd3(S), we examined factor binding to the ARG3
promoter, where transcriptional induction leads to NuA4 binding. Induction of arginine biosynthesis genes starts with binding of the Gcn4 activator, which then recruits NuA4 along with other coactivators (51
). Our ChIP assays show NuA4 binding concurrent with transcriptional activation. Interestingly, Rco1 is present at the promoter before induction but disappears as the gene is activated, as if NuA4 displaces Rpd3(S) from the promoter, thereby supporting our hypothesis of competition. We did not observe an increase in NuA4 binding at ARG3
in an rco1
mutant, possibly because NuA4 binding is dependent on other coactivators (51
). However, the defect in NuA4 binding at ARG3
in a pob3
mutant is partially suppressed deletion of the RCO1
gene. Also, of note, the presence of Rpd3(S) at the promoter of an uninduced gene is rather surprising, since previous work suggested Rpd3(S) is present only at the 3′ portion of actively transcribed regions (12
DNA damage results in the phosphorylation of the C-terminal tail of H2AX (or H2A in yeast), and this phosphorylation leads to the recruitment of multiple chromatin-modifying complexes, including Ino80, Swr1, and NuA4 (15
). The Arp4 subunit is required for efficient binding of NuA4 to nucleosomes with phosphorylated H2A(S129) (15
). We expressed the HO
endonuclease to induce double-strand breaks and found that NuA4 binding in the vicinity of the DNA breaks is significantly increased in an rco1
mutant lacking Rpd3(S). This increased NuA4 binding to double-strand breaks in an rco1
strain compared to that of the wild type strongly supports the idea of competition between NuA4 and Rpd3(S).
In response to DNA damage, the Mec1 and Tel1 kinases phosphorylate serine 129 of histone H2A (16
). An S129A mutation in histone H2A prevents this phosphorylation, and yeast strains with H2A(S129A) are sensitive to DNA-damaging agents (15
), possibly because NuA4 binds less efficiently to the regions of DNA damage. The fact that an rco1
mutation can suppress sensitivity of the H2A(S129A) mutant to DNA damage suggests that Rpd3(S) directly or indirectly inhibits binding of factors such as NuA4 that are important for repairing DNA damage.
The FACT complex plays an important role in DNA replication (6
), and our results suggest that the NuA4 KAT complex is also involved in DNA replication. A mutation in the Esa1 catalytic subunit results in mild sensitivity to HU, and an spt16 esa1
double mutant shows additivity in HU sensitivity. Importantly, the HU sensitivities of spt16
single mutants and the spt16 esa1
double mutant can be suppressed by disruption of RCO1
Our results suggest that NuA4 and FACT work together in promoting both transcription and DNA replication. FACT NuA4 double mutants show synthetic phenotypes, and a mutation in one reduces binding of the other factor. The two Rpd3 HDAC complexes differently affect this pathway, with Rpd3(L) acting in support and Rpd3(S) opposing the pathway. The synthetic defects seen in the Rpd3(L) FACT double mutants lead to the genetic argument that Rpd3(L) supports FACT; however, these synthetic defects can also be explained by increased levels of the Rpd3(S) complex in the sds3 mutant. This idea is supported by the observation that overexpression of the Rpd3(S)-specific Rco1 subunit is toxic in FACT mutants.
The competition between NuA4 and Rpd3(S) is apparent in cells with limited FACT activity, where optimal function becomes crucial for growth. FACT is stimulated by NuA4 and opposed by Rpd3(S), and further work is needed to understand how these enzymes that affect histone acetylation affect FACT activity.