SAGA Complex Research Articles

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

Elucidating the Role of Chromatin State and Transcription Factors on the Regulation of the Yeast Metabolic Cycle: A Multi-Omic Integrative Approach.

The Yeast Metabolic Cycle (YMC) is a model system in which levels of around 60% of the yeast transcripts cycle over time. The spatial and temporal resolution provided by the YMC has revealed that changes in the yeast metabolic landscape and chromatin status can be related to cycling gene expression. However, the interplay between histone modifications and transcription factor activity during the YMC is still poorly understood. Here we apply an innovative statistical approach to integrate chromatin state (ChIP-seq) and gene expression (RNA-seq) data to investigate the transcriptional control during the YMC. By using the multivariate regression models N-PLS (Partial Least Squares) and MORE (Multi-Omics REgulation) methodologies, we assessed the contribution of histone marks and transcription factors to the regulation of gene expression in the YMC. We found that H3K18ac and H3K9ac were the most important histone modifications, whereas Sfp1, Hfi1, Pip2, Mig2, and Yhp1 emerged as the most relevant transcription factors. A significant association in the co-regulation of gene expression was found between H3K18ac and the transcription factors Pip2 (involved in fatty acids metabolism), Xbp1 (cyclin implicated in the regulation of carbohydrate and amino acid metabolism), and Hfi1 (involved in the formation of the SAGA complex). These results evidence the crucial role of histone lysine acetylation levels in the regulation of gene expression in the YMC through the coordinated action of transcription factors and lysine acetyltransferases.

Epigenetic Factors and Mitochondrial Biology in Yeast: A New Paradigm for the Study of Cancer Metabolism?

Bidirectional cross-talk between nuclear and mitochondrial DNA is fundamental for cell homeostasis. Epigenetic mechanisms regulate the inter-organelle communication between nucleus and mitochondria. Recent research highlights not only the retrograde activation of nuclear gene transcription in case of mitochondria dysfunction, but also the role of post-translational modifications of mitochondrial proteins in respiratory metabolism. Here we discuss some aspects and novel findings in Saccharomyces cerevisiae. In yeast, KAT-Gcn5 and DUB-Ubp8 have a role in respiration and are localized, as single proteins, into mitochondria. These findings, beside the canonical and widely known nuclear activity of SAGA complex in chromatin regulation, provide novel clues on promising aspects linking evolutionary conserved epigenetic factors to the re-programmed metabolism of cancer cells.

Yeast two-hybrid screening reveals a dual function for the histone acetyltransferase GcnE by controlling glutamine synthesis and development in Aspergillus fumigatus.

The acetyltransferase GcnE is part of the SAGA complex which regulates fungal gene expression through acetylation of chromatin. Target genes of the histone acetyltransferase GcnE include those involved in secondary metabolism and asexual development. Here, we show that the absence of GcnE not only abrogated conidiation, but also strongly impeded vegetative growth of hyphae in the human pathogenic fungus Aspergillus fumigatus. A yeast two-hybrid screen using a Saccharomyces cerevisiae strain whose tRNA molecules were specifically adapted to express A. fumigatus proteins identified two unprecedented proteins that directly interact with GcnE. Glutamine synthetase GlnA as well as a hypothetical protein located on chromosome 8 (GbpA) were identified as binding partners of GcnE and their interaction was confirmed in vivo via bimolecular fluorescence complementation. Phenotypic characterization of gbpA and glnA deletion mutants revealed a role for GbpA during conidiogenesis and confirmed the central role of GlnA in glutamine biosynthesis. The increase of glutamine synthetase activity in the absence of GcnE indicated that GcnE silences GlnA through binding. This finding suggests an expansion of the regulatory role of GcnE in A. fumigatus.

Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures

SummaryDouble-strand breaks (DSBs) are extremely detrimental DNA lesions that can lead to cancer-driving mutations and translocations. Non-homologous end joining (NHEJ) and homologous recombination (HR) represent the two main repair pathways operating in the context of chromatin to ensure genome stability. Despite extensive efforts, our knowledge of DSB-induced chromatin still remains fragmented. Here, we describe the distribution of 20 chromatin features at multiple DSBs spread throughout the human genome using ChIP-seq. We provide the most comprehensive picture of the chromatin landscape set up at DSBs and identify NHEJ- and HR-specific chromatin events. This study revealed the existence of a DSB-induced monoubiquitination-to-acetylation switch on histone H2B lysine 120, likely mediated by the SAGA complex, as well as higher-order signaling at HR-repaired DSBs whereby histone H1 is evicted while ubiquitin and 53BP1 accumulate over the entire γH2AX domains.

Slt2 MAPK association with chromatin is required for transcriptional activation of Rlm1 dependent genes upon cell wall stress

The regulation of gene expression through the cell wall integrity (CWI) pathway in yeast is mainly coordinated by the MAPK Slt2 and the transcription factor Rlm1. In this work, we elucidate a new role for Slt2 as a part of the transcriptional activation machinery that regulates CWI gene expression in response to cell wall stress. We show that Slt2 is recruited to promoters and coding regions of CWI Rlm1-dependent genes in response to stress. This phenomenon is dependent both on the activation of the MAPK and its kinase activity. Slt2 binding is also dependent on Rlm1 and SWI/SNF and SAGA complexes. During the initial steps of transcription, the catalytic activity of Slt2 on Rlm1 is critical for the binding of the activator to promoters in response to stress. In addition, Slt2 itself acts as a transactivator, as it is able to induce the transcription of CWI responsive genes when it is bound to promoters through the Rlm1 binding domain independently of its catalytic activity. Slt2 interacts with RNA Pol II in a Rlm1-dependent manner to provide further support to a role of this MAPK as an integral component of the transcriptional complexes under cell wall stress. Selective recruitment and progression of the complex Slt2-RNA Pol II from the promoters to the coding regions of Rlm1-dependent genes does not rely on Paf1, suggesting a different mechanism from that which is exerted by Slt2 on the Swi4/Swi6 (SBF)-regulated genes.

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

Wheat microbiome bacteria can reduce virulence of a plant pathogenic fungus by altering histone acetylation

Interactions between bacteria and fungi have great environmental, medical, and agricultural importance, but the molecular mechanisms are largely unknown. Here, we study the interactions between the bacterium Pseudomonas piscium, from the wheat head microbiome, and the plant pathogenic fungus Fusarium graminearum. We show that a compound secreted by the bacteria (phenazine-1-carboxamide) directly affects the activity of fungal protein FgGcn5, a histone acetyltransferase of the SAGA complex. This leads to deregulation of histone acetylation at H2BK11, H3K14, H3K18, and H3K27 in F. graminearum, as well as suppression of fungal growth, virulence, and mycotoxin biosynthesis. Therefore, an antagonistic bacterium can inhibit growth and virulence of a plant pathogenic fungus by manipulating fungal histone modification.

The role of SAGA coactivator complex in snRNA transcription

ABSTRACT The general snRNA gene transcription apparatus has been extensively studied. However, the role of coactivators in this process is far from being clearly understood. Here, we have demonstrated that the Drosophila SAGA complex interacts with the PBP complex, the key component of the snRNA gene transcription apparatus, and is present at the promoter regions of the snRNA genes transcribed by both the RNA polymerase II and RNA polymerase III (U6 snRNA). We show that SAGA interacts with the Brf1 transcription factor, which is a part of the RNA polymerase III transcription apparatus and is present at promoters of a number of Pol III-transcribed genes. Mutations inactivating several SAGA subunit genes resulted in reduced snRNA levels in adult flies, indicating that SAGA is indeed the transcriptional coactivator for the snRNA genes. The transcription of the Pol II and Pol III-transcribed U genes was reduced by mutations in all tested SAGA complex subunits. Therefore, the transcription of the Pol II and Pol III-transcribed U genes was reduced by the mutations in the deubiquitinase module, as well as in the acetyltransferase module of the SAGA, indicating that the whole complex is essential for their transcription. Therefore, the SAGA complex activates snRNA genes suggesting its wide involvement in the regulation of gene transcription, and consequently, in the maintenance of cellular homeostasis.

Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration

Healthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae.

Abstract 363: Role of the SAGA histone acetylation module in lung cancer

Abstract Lung cancer causes the highest mortality in cancer-related deaths. Definition of novel molecular targets is needed since these cancers often become resistant to existing therapies. Epigenetic modifications may provide such targets. Recent reports suggest that the histone acetyltransferase (HAT) module within the transcriptional coactivator SAGA complex plays a role in cancer, creating a new link between epigenetic regulators and this disease. We are working to define the role of the SAGA HAT protein GCN5 in the regulation and function of oncogenes in lung cancers. Since our prior work indicates that GCN5 serves as a coactivator for MYC target genes, we hypothesize that inhibition of GCN5 will reduce lung oncogenesis by suppressing MYC functions. We also propose that inhibition of GCN5 will reduce lung cancer formation and progression in mouse models of lung cancer. Our preliminary data indicate that both GCN5 and MYC expression levels are upregulated and positively associated in mouse and human lung cancers. Both proteins are also associated with MYC target genes in lung cancer tissues. Inhibition of the GCN5 active site or bromodomain significantly (p < 0.05) reduces cell proliferation compared to normal lung epithelium cells. Studies investigating how GCN5 inhibition affects other lung cancer phenotypes are underway. Specific genes and pathways affected by GCN5 loss will be determined using RNA-Seq and RPPA assays to identify mechanisms behind observed phenotypes. In addition, we will determine whether knockout of Gcn5 reduces lung formation and progression in the KrasLA2 lung cancer mouse model. Our ongoing studies will determine whether targeting GCN5 represses multiple oncogenic pathways in cancer and whether it inhibits oncoproteins difficult to directly target, such as MYC. We expect that our findings will highlight the importance of epigenetic regulators in carcinogenesis and extend beyond our current research focus to other cancer types. Citation Format: Lisa Maria Mustachio, Jason Roszik, Sharon YR Dent. Role of the SAGA histone acetylation module in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 363.

The Pseudokinase Domain of Saccharomyces cerevisiae Tra1 Is Required for Nuclear Localization and Incorporation into the SAGA and NuA4 Complexes.

Tra1 is an essential component of the SAGA/SLIK and NuA4 complexes in S. cerevisiae, recruiting these co-activator complexes to specific promoters. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase (PI3K) domain. Unlike other PIKK family members (e.g., Tor1, Tor2, Mec1, Tel1), Tra1 has no demonstrable kinase activity. We identified three conserved arginine residues in Tra1 that reside proximal or within the cleft between the N- and C-terminal subdomains of the PI3K domain. To establish a function for Tra1’s PI3K domain and specifically the cleft region, we characterized a tra1 allele where these three arginine residues are mutated to glutamine. The half-life of the Tra1 protein is reduced but its steady state level is maintained at near wild-type levels by a transcriptional feedback mechanism. The tra1 allele results in slow growth under stress and alters the expression of genes also regulated by other components of the SAGA complex. Tra1 is less efficiently transported to the nucleus than the wild-type protein. Likely related to this, Tra1 associates poorly with SAGA/SLIK and NuA4. The ratio of Spt7SLIK to Spt7SAGA increases in the tra1 strain and truncated forms of Spt20 become apparent upon isolation of SAGA/SLIK. Intragenic suppressor mutations of tra1 map to the cleft region further emphasizing its importance. We propose that the PI3K domain of Tra1 is directly or indirectly important for incorporating Tra1 into SAGA and NuA4 and thus the biosynthesis and/or stability of the intact complexes.

Advances in Spt proteins and stress resistance of Saccharomyces cerevisiae

Spt proteins are defined as a large family of transcription regulators of the yeast Saccharomyces cerevisiae. They are crucial components of the SAGA complex that regulates transcription through interaction with the TATA box in the upstream region of the target genes. About 10% of total gene transcriptions are related to Spt proteins and these genes are highly related to environmental stress response. Such vast regulation network and complex mechanisms have become a hotspot. Spt proteins are also important to suppress transposon-induced mutations, being a switch on regulation of transposon behaviors and adaptive evolution of Saccharomyces cerevisiae. Besides that, some Spt proteins are directly involved in regulating unsaturated lipid acids synthesis, which could remold the cell membrane to resist environmental stresses. Here, we review Spt proteins, the advances in Spt proteins study, and their potential applications in improving yeast's stress resistance through transcription regulation, transposon activity regulation and cell membrane alternation.

The Adaptor Protein ENY2 Is a Component of the Deubiquitination Module of the Arabidopsis SAGA Transcriptional Co-activator Complex but not of the TREX-2 Complex

The conserved nuclear protein ENY2 (Sus1 in yeast) is involved in coupling transcription and mRNA export in yeast and metazoa, as it is a component both of the transcriptional co-activator complex SAGA and of the mRNA export complex TREX-2. Arabidopsis thaliana ENY2 is widely expressed in the plant and it localizes to the nucleoplasm, but unlike its yeast/metazoan orthologs, it is not enriched in the nuclear envelope. Affinity purification of ENY2 in combination with mass spectrometry revealed that it co-purified with SAGA components, but not with the nuclear pore-associated TREX-2. In addition, further targeted proteomics analyses by reciprocal tagging established the composition of the Arabidopsis SAGA complex consisting of the four modules HATm, SPTm, TAFm and DUBm, and that several SAGA subunits occur in alternative variants. While the HATm, SPTm and TAFm robustly co-purified with each other, the deubiquitination module (DUBm) appears to associate with the other SAGA modules more weakly/dynamically. Consistent with a homology model of the Arabidopsis DUBm, the SGF11 protein interacts directly with ENY2 and UBP22. Plants depleted in the DUBm components, SGF11 or ENY2, are phenotypically only mildly affected, but they contain increased levels of ubiquitinated histone H2B, indicating that the SAGA-DUBm has histone deubiquitination activity in plants. In addition to transcription-related proteins (i.e., transcript elongation factors, Mediator), many splicing factors were found to associate with SAGA, linking the SAGA complex and ongoing transcription with mRNA processing.

Mutational analysis of TAF6 revealed the essential requirement of the histone-fold domain and the HEAT repeat domain for transcriptional activation.

TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex invivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.

The H2B deubiquitinase Usp22 promotes antibody class switch recombination by facilitating non-homologous end joining

Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.

Whole-Genome Sequencing of Suppressor DNA Mixtures Identifies Pathways That Compensate for Chromosome Segregation Defects in Schizosaccharomyces pombe.

Suppressor screening is a powerful method to identify genes that, when mutated, rescue the temperature sensitivity of the original mutation. Previously, however, identification of suppressor mutations has been technically difficult. Due to the small genome size of Schizosaccharomyces pombe, we developed a spontaneous suppressor screening technique, followed by a cost-effective sequencing method. Genomic DNAs of 10 revertants that survived at the restrictive temperature of the original temperature sensitive (ts) mutant were mixed together as one sample before constructing a library for sequencing. Responsible suppressor mutations were identified bioinformatically based on allele frequency. Then, we isolated a large number of spontaneous extragenic suppressors for three ts mutants that exhibited defects in chromosome segregation at their restrictive temperature. Screening provided new insight into mechanisms of chromosome segregation: loss of Ufd2 E4 multi-ubiquitination activity suppresses defects of an AAA ATPase, Cdc48. Loss of Wpl1, a releaser of cohesin, compensates for the Eso1 mutation, which may destabilize sister chromatid cohesion. The segregation defect of a ts histone H2B mutant is rescued if it fails to be deubiquitinated by the SAGA complex, because H2B is stabilized by monoubiquitination.

Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling.

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high-affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID-dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

Diversification of the Histone Acetyltransferase GCN5 through Alternative Splicing in Brachypodium distachyon.

The epigenetic modulatory SAGA complex is involved in various developmental and stress responsive pathways in plants. Alternative transcripts of the SAGA complex's enzymatic subunit GCN5 have been identified in Brachypodium distachyon. These splice variants differ based on the presence and integrity of their conserved domain sequences: the histone acetyltransferase domain, responsible for catalytic activity, and the bromodomain, involved in acetyl-lysine binding and genomic loci targeting. GCN5 is the wild-type transcript, while alternative splice sites result in the following transcriptional variants: L-GCN5, which is missing the bromodomain and S-GCN5, which lacks the bromodomain as well as certain motifs of the histone acetyltransferase domain. Absolute mRNA quantification revealed that, across eight B. distachyon accessions, GCN5 was the dominant transcript isoform, accounting for up to 90% of the entire transcript pool, followed by L-GCN5 and S-GCN5. A cycloheximide treatment further revealed that the S-GCN5 splice variant was degraded through the nonsense-mediated decay pathway. All alternative BdGCN5 transcripts displayed similar transcript profiles, being induced during early exposure to heat and displaying higher levels of accumulation in the crown, compared to aerial tissues. All predicted protein isoforms localize to the nucleus, which lends weight to their purported epigenetic functions. S-GCN5 was incapable of forming an in vivo protein interaction with ADA2, the transcriptional adaptor that links the histone acetyltransferase subunit to the SAGA complex, while both GCN5 and L-GCN5 interacted with ADA2, which suggests that a complete histone acetyltransferase domain is required for BdGCN5-BdADA2 interaction in vivo. Thus, there has been a diversification in BdGCN5 through alternative splicing that has resulted in differences in conserved domain composition, transcript fate and in vivo protein interaction partners. Furthermore, our results suggest that B. distachyon may harbor compositionally distinct SAGA-like complexes that differ based on their histone acetyltransferase subunit.

Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes.

General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA- and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes.