Stéphane Allard

NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM‐related cofactor Tra1p

Post‐translational acetylation of histone H4 N‐terminal tail in chromatin has been associated with several nuclear processes including transcription. We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4. NuA4 has an apparent molecular mass of 1.3 MDa. All four conserved lysines of histone H4 can be acetylated by NuA4. We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast. Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature‐sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature. Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM‐related essential gene homologous to human TRRAP, an essential cofactor for c‐Myc‐ and E2F‐mediated oncogenic transformation. Finally, the ability of NuA4 to stimulate GAL4–VP16‐driven transcription from chromatin templates in vitro is also lost in the temperature‐sensitive esa1 mutant. The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator‐interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control.

Multiple Links between the NuA4 Histone Acetyltransferase Complex and Epigenetic Control of Transcription

NuA4 is an essential histone H4/H2A acetyltransferase complex that interacts with activators and stimulates transcription in vitro. We have identified three novel NuA4 subunits: Act3/Arp4, an actin-related protein implicated in epigenetic control of transcription, Act1, and Epl1, a protein homologous to Drosophila Enhancer of Polycomb. Act3/Arp4 binds nucleosomes in vitro and is required for NuA4 integrity in vivo. Mutations in ACT3 and acetyltransferase-encoding ESA1 cause gene-specific transcription defects. Accordingly, NuA4 is localized in precise loci within the nucleus and does not overlap with the silent chromatin marker Sir3. These data along with the known epigenetic roles of Act3/Arp4 and homologs of Epl1 and Esa1 strongly support an essential role for chromatin structure modification by NuA4 in transcription regulation in vivo.

Role of Dot1-Dependent Histone H3 Methylation in G1 and S Phase DNA Damage Checkpoint Functions of Rad9

ABSTRACT We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Δ) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Δ checkpoint defects. Whereas dot1Δ alone confers minimal DNA damage sensitivity, combining dot1Δ with histone methyltransferase mutations set1Δ and set2Δ markedly enhances lethality. Interestingly, set1Δ and set2Δ mutants remain G1 checkpoint competent, but set1Δ displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Δ checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.

Regulation of NuA4 Histone Acetyltransferase Activity in Transcription and DNA Repair by Phosphorylation of Histone H4

ABSTRACT The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

Eaf1 Is the Platform for NuA4 Molecular Assembly That Evolutionarily Links Chromatin Acetylation to ATP-Dependent Exchange of Histone H2A Variants

ABSTRACT Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells.

Role of an ING1 Growth Regulator in Transcriptional Activation and Targeted Histone Acetylation by the NuA4 Complex

ABSTRACT The yeast NuA4 complex is a histone H4 and H2A acetyltransferase involved in transcription regulation and essential for cell cycle progression. We identify here a novel subunit of the complex, Yng2p, a plant homeodomain (PHD)-finger protein homologous to human p33/ING1, which has tumor suppressor activity and is essential for p53 function. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable stoichiometric association of this protein with purified NuA4. Yeast cells harboring a deletion of theYNG2 gene show severe growth phenotype and have gene-specific transcription defects. NuA4 complex purified from the mutant strain is low in abundance and shows weak histone acetyltransferase activity. We demonstrate conservation of function by the requirement of Yng2p for p53 to function as a transcriptional activator in yeast. Accordingly, p53 interacts with NuA4 in vitro and in vivo, an interaction reminiscent of the p53-ING1 physical link in human cells. The growth defect of Δyng2 cells can be rescued by the N-terminal part of the protein, lacking the PHD-finger. While Yng2 PHD-finger is not required for p53 interaction, it is necessary for full expression of the p53-responsive gene and other NuA4 target genes. Transcriptional activation by p53 in vivo is associated with targeted NuA4-dependent histone H4 hyperacetylation, while histone H3 acetylation levels remain unchanged. These results emphasize the essential role of the NuA4 complex in the control of cell proliferation through gene-specific transcription regulation. They also suggest that regulation of mammalian cell proliferation by p53-dependent transcriptional activation functions through recruitment of an ING1-containing histone acetyltransferase complex.

Recruitment of the NuA4 complex poises the PHO5 promoter for chromatin remodeling and activation

The remodeling of the promoter chromatin structure is a key event for the induction of the PHO5 gene. Two DNA‐binding proteins Pho2 and Pho4 are critical for this step. We found that the NuA4 histone acetyltransferase complex is essential for PHO5 transcriptional induction without affecting Pho4 translocation upon phosphate starvation. Our data also indicate that NuA4 is critical for the chromatin remodeling event that occurs over the PHO5 promoter prior to activation. Using Chromatin IP analysis, we found that Esa1‐dependent histone H4 acetylation at the PHO5 promoter correlates with specific recruitment of the NuA4 complex to this locus under repressing conditions. We demonstrate that the homeodomain transcriptional activator Pho2 is responsible for this recruitment in vivo and interacts directly with the NuA4 complex. Finally, we show that Pho4 is unable to bind the PHO5 promoter without prior action of NuA4. These results indicate that, before induction, NuA4 complex recruitment by Pho2 is an essential event that presets the PHO5 promoter for subsequent binding by Pho4, chromatin remodeling and transcription.

Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes.

We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4–NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo–NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4–NCP interactions.

The Candida glabrata Amt1 copper-sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification

The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper‐inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome. Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE. In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels. We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific. Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE. Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation.