Amine Nourani

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.

Histone Chaperones: Modulators of Chromatin Marks

The many factors that control chromatin biology play key roles in essential nuclear functions like transcription, DNA damage response and repair, recombination, and replication and are critical for proper cell-cycle progression, stem cell renewal, differentiation, and development. These players belong to four broad classes: histone modifiers, chromatin remodelers, histone variants, and histone chaperones. A large number of studies have established the existence of an intricate functional crosstalk between the different factors, not only within a single class but also between different classes. In light of this, while many recent reviews have focused on structure and functions of histone chaperones, the current text highlights novel and striking links that have been established between these proteins and posttranslational modifications of histones and discusses the functional consequences of this crosstalk. These findings feed a current hot question of how cell memory may be maintained through epigenetic mechanisms involving histone chaperones.

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.

Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae

ABSTRACT Spt2/Sin1 is a DNA binding protein with HMG-like domains that has been suggested to play a role in chromatin-mediated transcription in Saccharomyces cerevisiae. Previous studies have suggested models in which Spt2 plays an inhibitory role in the initiation of transcription of certain genes. In this work, we have taken several approaches to study Spt2 in greater detail. Our results have identified previously unknown genetic interactions between spt2Δ and mutations in genes encoding transcription elongation factors, including members of the PAF and HIR/HPC complexes. In addition, genome-wide and gene-specific chromatin immunoprecipitation analyses suggest that Spt2 is primarily associated with coding regions in a transcription-dependent fashion. Furthermore, our results show that Spt2, like other elongation factors, is required for the repression of transcription from a cryptic promoter within a coding region and that Spt2 is also required for repression of recombination within transcribed regions. Finally, we provide evidence that Spt2 plays a role in regulating the levels of histone H3 over transcribed regions. Taken together, our results suggest a direct link for Spt2 with transcription elongation, chromatin dynamics, and genome stability.

The Rtt106 histone chaperone is functionally linked to transcription elongation and is involved in the regulation of spurious transcription from cryptic promoters in yeast.

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106Δ and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.

Transcription regulation by the noncoding RNA SRG1 requires Spt2-dependent chromatin deposition in the wake of RNA polymerase II.

ABSTRACT Spt2 is a chromatin component with roles in transcription and posttranscriptional regulation. Recently, we found that Spt2 travels with RNA polymerase II (RNAP II), is involved in elongation, and plays important roles in chromatin modulations associated with this process. In this work, we dissect the function of Spt2 in the repression of SER3. This gene is repressed by a transcription interference mechanism involving the transcription of an adjacent intergenic region, SRG1, that leads to the production of a noncoding RNA (ncRNA). We find that Spt2 and Spt6 are required for the repression of SER3 by SRG1 transcription. Intriguingly, we demonstrate that these effects are not mediated through modulations of the SRG1 transcription rate. Instead, we show that the SRG1 region overlapping the SER3 promoter is occluded by randomly positioned nucleosomes that are deposited behind RNAP II transcribing SRG1 and that their deposition is dependent on the presence of Spt2. Our data indicate that Spt2 is required for the major chromatin deposition pathway that uses old histones to refold nucleosomes in the wake of RNAP II at the SRG1-SER3 locus. Altogether, these observations suggest a new mechanism of repression by ncRNA transcription involving a repressive nucleosomal structure produced by an Spt2-dependent pathway following RNAP II passage.

Evidence that the Localization of the Elongation Factor Spt16 Across Transcribed Genes Is Dependent Upon Histone H3 Integrity in Saccharomyces cerevisiae

A previous study of histone H3 in Saccharomyces cerevisiae identified a mutant with a single amino acid change, leucine 61 to tryptophan, that confers several transcriptional defects. We now present several lines of evidence that this H3 mutant, H3-L61W, is impaired at the level of transcription elongation, likely by altered interactions with the conserved factor Spt16, a subunit of the transcription elongation complex yFACT. First, a selection for suppressors of the H3-L61W cold-sensitive phenotype has identified novel mutations in the gene encoding Spt16. These genetic interactions are allele specific, suggesting a direct interaction between H3 and Spt16. Second, similar to several other elongation and chromatin mutants, including spt16 mutants, an H3-L61W mutant allows transcription from a cryptic promoter within the FLO8 coding region. Finally, chromatin-immunoprecipitation experiments show that in an H3-L61W mutant there is a dramatically altered profile of Spt16 association over transcribed regions, with reduced levels over 5′-coding regions and elevated levels over the 3′ regions. Taken together, these and other results provide strong evidence that the integrity of histone H3 is crucial for ensuring proper distribution of Spt16 across transcribed genes and suggest a model for the mechanism by which Spt16 normally dissociates from DNA following transcription.

Eaf5/7/3 form a functionally independent NuA4 submodule linked to RNA polymerase II‐coupled nucleosome recycling

The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13‐subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3‐binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene‐specific and genome‐wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replication‐independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.