Rhea T. Utley

Transcriptional activators direct histone acetyltransferase complexes to nucleosomes

Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5,, as well as human p300/CBP,, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4–VP16-bound nucleosomes for acetylation. We find that Gal4–VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

The diverse functions of histone acetyltransferase complexes

Although histone acetylation has historically been linked to transcription activation, recent studies indicate that this modification and the enzymes that catalyze it have much broader and diverse functions. Histone acetyltransferase complexes are involved in such diverse processes as transcription activation, gene silencing, DNA repair and cell-cycle progression. The high conservation of the acetyltransferase complexes and their functions illustrates their central role in cell growth and development.

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.

Persistent Site-Specific Remodeling of a Nucleosome Array by Transient Action of the SWI/SNF Complex

The SWI/SNF complex participates in the restructuring of chromatin for transcription. The function of the yeast SWI/SNF complex in the remodeling of a nucleosome array has now been analyzed in vitro. Binding of the purified SWI/SNF complex to a nucleosome array disrupted multiple nucleosomes in an adenosine triphosphate-dependent reaction. However, removal of SWI/SNF left a deoxyribonuclease I-hypersensitive site specifically at a nucleosome that was bound by derivatives of the transcription factor Gal4p. Analysis of individual nucleosomes revealed that the SWI/SNF complex catalyzed eviction of histones from the Gal4-bound nucleosomes. Thus, the transient action of the SWI/SNF complex facilitated irreversible disruption of transcription factor-bound nucleosomes.

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.

NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex

Structural and functional analyses of nucleosomes containing histone variant H2A.Z have drawn a lot of interest over the past few years. Important work in budding yeast has shown that H2A.Z (Htz1)-containing nucleosomes are specifically located on the promoter regions of genes, creating a specific chromatin structure that is poised for disassembly during transcription activation. The SWR1 complex is responsible for incorporation of Htz1 into nucleosomes through ATP-dependent exchange of canonical H2A-H2B dimers for Htz1-H2B dimers. Interestingly, the yeast SWR1 complex is functionally linked to the NuA4 acetyltransferase complex in vivo. NuA4 and SWR1 are physically associated in higher eukaryotes as they are homologous to the TIP60/p400 complex, which encompasses both histone acetyltransferase (Tip60) and histone exchange (p400/Domino) activities. Here we present work investigating the impact of NuA4-dependent acetylation on SWR1-driven incorporation of H2A.Z into chromatin. Using in vitro histone exchange assays with native chromatin, we demonstrate that prior chromatin acetylation by NuA4 greatly stimulates the exchange of H2A for H2A.Z. Interestingly, we find that acetylation of H2A or H4 N-terminal tails by NuA4 can independently stimulate SWR1 activity. Accordingly, we demonstrate that mutations of H4 or H2A N-terminal lysine residues have similar effects on H2A.Z incorporation in vivo, and cells carrying mutations in both tails are nonviable. Finally, depletion experiments indicate that the bromodomain-containing protein Bdf1 is important for NuA4-dependent stimulation of SWR1. These results provide important mechanistic insight into the functional cross-talk between chromatin acetylation and ATP-dependent exchange of histone H2A variants.

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.

Differential repression of transcription factor binding by histone H1 is regulated by the core histone amino termini.

In order to investigate the interrelated roles of nucleosome cores and histone H1 in transcription repression, we have employed a purified system to analyze the function of H1 in the repression of transcription factor binding to nucleosomes. H1 binding to nucleosome cores resulted in the repression of USF binding to nucleosomes. By contrast, H1 only slightly inhibited the binding of GAL4‐AH, indicating that H1 differentially represses the binding of factors with different DNA‐binding domains. H1‐mediated repression of factor binding was dependent on the core histone amino‐terminal tails. Removal of these domains alleviated H1‐mediated repression and increased acetylation of these domains partly alleviated repression by H1. H1 binding assays suggest a less stable interaction of histone H1 with the core particle in the absence of the amino termini.