Spt-Ada-Gcn5 Acetyltransferase Research Articles

Progressive degeneration in a new Drosophila model of spinocerebellar ataxia type 7

Spinocerebellar ataxia type 7 (SCA7) is a progressive neurodegenerative disorder resulting from abnormal expansion of an uninterrupted polyglutamine (polyQ) repeat in its disease protein, ataxin-7 (ATXN7). ATXN7 is part of Spt-Ada-Gcn5 acetyltransferase (SAGA), an evolutionarily conserved transcriptional coactivation complex with critical roles in chromatin remodeling, cell signaling, neurodifferentiation, mitochondrial health and autophagy. SCA7 is dominantly inherited and characterized by genetic anticipation and high repeat-length instability. Patients with SCA7 experience progressive ataxia, atrophy, spasticity, and blindness. There is currently no cure for SCA7, and therapies are aimed at alleviating symptoms to increase quality of life. Here, we report novel Drosophila lines of SCA7 with polyQ repeats in wild-type and human disease patient range. We find that ATXN7 expression has age- and polyQ repeat length-dependent reduction in fruit fly survival and retinal instability, concomitant with increased ATXN7 protein aggregation. These new lines will provide important insight on disease progression that can be used in the future to identify therapeutic targets for SCA7 patients.

The KAT module of the SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma.

Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.

H2B oncohistones cause homologous recombination defect and genomic instability through reducing H2B monoubiquitination in Schizosaccharomyces pombe

Canonical oncohistones are histone H3 mutations in the N-terminal tail associated with tumors and affect gene expression by altering H3 post-translational modifications (PTMs) and the epigenetic landscape. Noncanonical oncohistone mutations occur in both tails and globular domains of all 4 core histones and alter gene expression by perturbing chromatin remodeling. However, the effects and mechanisms of noncanonical oncohistones remain largely unknown. Here we characterized 16 noncanonical H2B oncohistones in the fission yeast Schizosaccharomyces pombe. We found that 7 of them exhibited temperature sensitivities and 11 exhibited genotoxic sensitivities. A detailed study of 2 of these onco-mutants H2BG52D and H2BP102L revealed that they were defective in homologous recombination (HR) repair with compromised histone eviction and Rad51 recruitment. Interestingly, their genotoxic sensitivities and HR defects were rescued by inactivation of the H2BK119 deubiquitination function of Ubp8 in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. The levels of H2BK119 monoubiquitination (H2Bub) in the H2BG52D and H2BP102L mutants are reduced in global and local DNA break sites presumably due to enhanced recruitment of Ubp8 onto nucleosomes, and are recovered upon loss of H2B deubiquitination function of the SAGA complex. Moreover, H2BG52D and H2BP102L heterozygotes exhibit genotoxic sensitivities and reduced H2Bub in cis. We therefore conclude that H2BG52D and H2BP102L oncohistones affect HR repair and genome stability via reduction of H2Bub and propose that other noncanonical oncohistones may also affect histone PTMs to cause diseases.

Identification of banana ADA1 gene family members and their expression profiles under biotic and abiotic stresses

The Spt-Ada-Gcn5-acetyltransferase (SAGA) is an ancillary transcription initiation complex which is highly conserved. The ADA1 (alteration/deficiency in activation 1, also called histone H2A functional interactor 1, HFI1) is a subunit in the core module of the SAGA protein complex. ADA1 plays an important role in plant growth and development as well as stress resistance. In this paper, we performed genome-wide identification of banana ADA1 gene family members based on banana genomic data, and analyzed the basic physicochemical properties, evolutionary relationships, selection pressure, promoter cis-acting elements, and its expression profiles under biotic and abiotic stresses. The results showed that there were 10, 6, and 7 family members in Musa acuminata, Musa balbisiana and Musa itinerans. The members were all unstable and hydrophilic proteins, and only contained the conservative SAGA-Tad1 domain. Both MaADA1 and MbADA1 have interactive relationship with Sgf11 (SAGA-associated factor 11) of core module in SAGA. Phylogenetic analysis revealed that banana ADA1 gene family members could be divided into 3 classes. The evolution of ADA1 gene family members was mostly influenced by purifying selection. There were large differences among the gene structure of banana ADA1 gene family members. ADA1 gene family members contained plenty of hormonal elements. MaADA1-1 may play a prominent role in the resistance of banana to cold stress, while MaADA1 may respond to the Panama disease of banana. In conclusion, this study suggested ADA1 gene family members are highly conserved in banana, and may respond to biotic and abiotic stress.

USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of function impacts MMTV promoter activity.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)-driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.

The transcription factor FgStuA regulates virulence and mycotoxin biosynthesis via recruiting the SAGA complex in Fusarium graminearum.

The conserved Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex controls eukaryotic transcription by modifying acetylation of histones. However, the mechanisms for this complex in regulating the transcription of target-specific genes remain largely unknown in phytopathogenic fungi. A filamentous fungal-specific transcription factor FgStuA was identified to interact with the SAGA complex physically. The coordinative mechanisms of FgStuA with the SAGA complex in regulating secondary metabolism and virulence were investigated in Fusarium graminearum with genetic, biochemical and molecular techniques. The transcription factor FgStuA binds to a 7-bp cis-element (BVTGCAK) of its target gene promoter. Under mycotoxin deoxynivalenol (DON) induction conditions, FgStuA recruits the SAGA complex into the promoter of TRI6, a core regulator of the DON biosynthesis gene cluster, leading to enhanced transcription of TRI6. During this process, we found that FgStuA is subject to acetylation by the SAGA complex, and acetylation of FgStuA plays a critical role for its enrichment in the TRI6 promoter. In addition, FgStuA together with the SAGA complex modulates fungal virulence. This study uncovers a novel regulatory mechanism of a transcription factor, which recruits and interacts with the SAGA complex to activate specific gene expression in pathogenic fungi.

ATAC and SAGA co-activator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct

To understand the function of multisubunit complexes, it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here, we demonstrate that the core modules of ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription co-activator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, a SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histone proteins. In contrast, ATAC complex subunits cannot be detected in the cytoplasm of mammalian cells. However, an endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble using co-translational pathways, but their subcellular localization, cytoplasmic abundance, and functions are distinct.

Differing SAGA module requirements for NCR-sensitive gene transcription in yeast.

Nitrogen catabolite repression (NCR) is a means for yeast to adapt its transcriptome to changing nitrogen sources in its environment. In conditions of derepression (under poor nitrogen conditions, upon rapamycin treatment, or when glutamine production is inhibited), two transcriptional activators of the GATA family are recruited to NCR-sensitive promoters and activate transcription of NCR-sensitive genes. Earlier observations have involved the Spt-Ada-Gcn5 acetyltransferase(SAGA) chromatin remodeling complex in these transcriptional regulations. In this report, we provide an illustration of the varying NCR-sensitive responses and question whether differing SAGA recruitment could explain this diversity of responses.

A novel ubiquitin-proteasome system regulation of Sgf73/ataxin-7 that maintains the integrity of the coactivator SAGA in orchestrating transcription.

Ataxin-7 maintains the integrity of Spt-Ada-Gcn5-Acetyltransferase (SAGA), an evolutionarily conserved coactivator in stimulating preinitiation complex (PIC) formation for transcription initiation, and thus, its upregulation or downregulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7's yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73's abundance, which enhances recruitment of TATA box-binding protein (TBP) (that nucleates PIC formation) to the promoter but impairs transcription elongation. Further, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7's abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health and implicate alteration of such regulation in diseases.

Spt3 and Spt8 Are Involved in the Formation of a Silencing Boundary by Interacting with TATA-Binding Protein

In Saccharomyces cerevisiae, a heterochromatin-like chromatin structure called the silencing region is present at the telomere as a complex of Sir2, Sir3, and Sir4. Although spreading of the silencing region is blocked by histone acetylase-mediated boundary formation, the details of the factors and mechanisms involved in the spread and formation of the boundary at each telomere are unknown. Here, we show that Spt3 and Spt8 block the spread of the silencing regions. Spt3 and Spt8 are members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, which has histone acetyltransferase activity. We performed microarray analysis of the transcriptome of spt3Δ and spt8Δ strains and RT-qPCR analysis of the transcript levels of genes from the subtelomeric region in mutants in which the interaction of Spt3 with TATA-binding protein (TBP) is altered. The results not only indicated that both Spt3 and Spt8 are involved in TBP-mediated boundary formation on the right arm of chromosome III, but also that boundary formation in this region is DNA sequence independent. Although both Spt3 and Spt8 interact with TBP, Spt3 had a greater effect on genome-wide transcription. Mutant analysis showed that the interaction between Spt3 and TBP plays an important role in the boundary formation.

The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.

The Gcn5-Ada2-Ada3 histone acetyltransferase module has divergent roles in pathogenesis of Candida glabrata.

Candida glabrata is an opportunistic fungal pathogen and the second most prevalent species isolated from candidiasis patients. C. glabrata has intrinsic tolerance to antifungal drugs and oxidative stresses and the ability to adhere to mucocutaneous surfaces. However, knowledge about the regulation of its virulence traits is limited. The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex modulates gene transcription by histone acetylation through the histone acetyltransferase (HAT) module comprised of Gcn5-Ada2-Ada3. Previously, we showed that the ada2 mutant was hypervirulent but displayed decreased tolerance to antifungal drugs and cell wall perturbing agents. In this study, we further characterized the functions of Ada3 and Gcn5 in C. glabrata. We found that single, double, or triple deletions of the HAT module, as expected, resulted in a decreased level of acetylation on histone H3 lysine 9 (H3K9) and defective growth. These mutants were more susceptible to antifungal drugs, oxidative stresses, and cell wall perturbing agents compared with the wild-type. In addition, HAT module mutants exhibited enhanced agar invasion and upregulation of adhesin and proteases encoding genes, whereas the biofilm formation of those mutants was impaired. Interestingly, HAT module mutants exhibited enhanced induction of catalases (CTA1) expression upon treatment with H2O2 compared with the wild-type. Lastly, although ada3 and gcn5 exhibited marginal hypervirulence, the HAT double and triple mutants were hypervirulent in a murine model of candidiasis. In conclusion, the HAT module of the SAGA complex plays unique roles in H3K9 acetylation, drug tolerance, oxidative stress response, adherence, and virulence in C. glabrata.

Abstract IA004: Novel inhibitors of histone H2B deubiquitinating enzymes

Abstract Histone H2B-K120 is dynamically ubiquitinated and deubiquitinated during transcription and is a hallmark of actively transcribed genes. The Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator contains a four-protein subcomplex called the deubiquitinating enzyme (DUB) module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit ubiquitin-specific protease 22 (USP22), which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 compared with a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for drug development. Citation Format: Cynthia Wolberger. Novel inhibitors of histone H2B deubiquitinating enzymes. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA004.

Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia

Abstract Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting >600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks. Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR004.

THE SAGA COMPLEX IS ESSENTIAL FOR THE REGULATION OF GENES INVOLVED IN YEAST TREHALOSE METABOLISM

Saccharomyces cerevisiae accumulates trehalose as a stress metabolite in adverse environmental conditions. The trehalose synthesis and breakdown are important for the regulation of trehalose levels within the yeast cell. Therefore, TPS1 and NTH1 gene expressions are tightly regulated during transcription and also translation. Since both genes contain Stress Response Elements (STRE) in the promoter regions, they are co-activated under stress conditions. However, the presence of similar regulatory elements in the promoter of both genes shows that these genes undergo a different regulation at the transcriptional level. In our study, the role of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in the transcriptional regulation of TPS1 and NTH1 genes was determined in nutrient-poor environment. For that purpose, the wild type and Δada1 mutant yeast cells, where Ada1p is a member of the SAGA complex, were grown in normal and nitrogen starvation conditions. In addition, trehalose level was detected enzymatically in both wild type and mutant yeast cells. In silico promoter analysis of TPS1 and NTH1 promoters revealed that the STRE sequences required for binding of Msn2/4 transcription factors are closed by nucleosomes at the NTH1 promoter, but open at the TPS1 promoter. In the absence of Ada1p, stress-induced promoter activation in the TPS1 gene was observed, while NTH1 gene expression was not activated. According to these results, the nucleosomes spanning the STRE sequences could not be mobilized in the absence of Ada1 protein, and therefore the Msn2/4 transcription factors cannot bind to the promoter and activate the NTH1 gene expression under stress conditions. It was also observed that in the absence of Ada1p, trehalose accumulation was reduced regardless of stress conditions.

SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells.

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone-fold domain (HFD)-containing proteins forming three histone-fold (HF) pairs, to which the double HFD-containing SUPT3H adds one HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that (i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H, (ii) SUPT3H is not essential for mESC survival, but required for their growth and self-renewal, and (iii) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

Conformational landscape of the yeast SAGA complex as revealed by cryo-EM

Spt-Ada-Gcn5-Acetyltransferase (SAGA) is a conserved multi-subunit complex that activates RNA polymerase II-mediated transcription by acetylating and deubiquitinating nucleosomal histones and by recruiting TATA box binding protein (TBP) to DNA. The prototypical yeast Saccharomyces cerevisiae SAGA contains 19 subunits that are organized into Tra1, core, histone acetyltransferase, and deubiquitination modules. Recent cryo-electron microscopy studies have generated high-resolution structural information on the Tra1 and core modules of yeast SAGA. However, the two catalytical modules were poorly resolved due to conformational flexibility of the full assembly. Furthermore, the high sample requirement created a formidable barrier to further structural investigations of SAGA. Here, we report a workflow for isolating/stabilizing yeast SAGA and preparing cryo-EM specimens at low protein concentration using a graphene oxide support layer. With this procedure, we were able to determine a cryo-EM reconstruction of yeast SAGA at 3.1 Å resolution and examine its conformational landscape with the neural network-based algorithm cryoDRGN. Our analysis revealed that SAGA adopts a range of conformations with its HAT module and central core in different orientations relative to Tra1.

SAGA-Dependent Histone H2Bub1 Deubiquitination Is Essential for Cellular Ubiquitin Balance during Embryonic Development.

Ubiquitin (ub) is a small, highly conserved protein widely expressed in eukaryotic cells. Ubiquitination is a post-translational modification catalyzed by enzymes that activate, conjugate, and ligate ub to proteins. Substrates can be modified either by addition of a single ubiquitin molecule (monoubiquitination), or by conjugation of several ubs (polyubiquitination). Monoubiquitination acts as a signaling mark to control diverse biological processes. The cellular and spatial distribution of ub is determined by the opposing activities of ub ligase enzymes, and deubiquitinases (DUBs), which remove ub from proteins to generate free ub. In mammalian cells, 1–2% of total histone H2B is monoubiquitinated. The SAGA (Spt Ada Gcn5 Acetyl-transferase) is a transcriptional coactivator and its DUB module removes ub from H2Bub1. The mammalian SAGA DUB module has four subunits, ATXN7, ATXN7L3, USP22, and ENY2. Atxn7l3−/− mouse embryos, lacking DUB activity, have a five-fold increase in H2Bub1 retention, and die at mid-gestation. Interestingly, embryos lacking the ub encoding gene, Ubc, have a similar phenotype. Here we provide a current overview of data suggesting that H2Bub1 retention on the chromatin in Atxn7l3−/− embryos may lead to an imbalance in free ub distribution. Thus, we speculate that ATXN7L3-containing DUBs impact the free cellular ub pool during development.

Sus1 maintains a normal lifespan through regulation of TREX-2 complex-mediated mRNA export.

Eukaryotic gene expression requires multiple cellular events, including transcription and RNA processing and transport. Sus1, a common subunit in both the Spt-Ada-Gcn5 acetyltransferase (SAGA) and transcription and export complex-2 (TREX-2) complexes, is a key factor in coupling transcription activation to mRNA nuclear export. Here, we report that the SAGA DUB module and TREX-2 distinctly regulate yeast replicative lifespan in a Sir2-dependent and -independent manner, respectively. The growth and lifespan impaired by SUS1 loss depend on TREX-2 but not on the SAGA DUB module. Notably, an increased dose of the mRNA export factors Mex67 and Dbp5 rescues the growth defect, shortened lifespan, and nuclear accumulation of poly(A)+ RNA in sus1Δ cells, suggesting that boosting the mRNA export process restores the mRNA transport defect and the growth and lifespan damage in sus1Δ cells. Moreover, Sus1 is required for the proper association of Mex67 and Dbp5 with the nuclear rim. Together, these data indicate that Sus1 links transcription and mRNA nuclear export to the lifespan control pathway, suggesting that prevention of an abnormal accumulation of nuclear RNA is necessary for maintenance of a normal lifespan.

Inhibition of histone acetyltransferase GCN5 by a transcription factor FgPacC controls fungal adaption to host-derived iron stress.

Poaceae plants can locally accumulate iron to suppress pathogen infection. It remains unknown how pathogens overcome host-derived iron stress during their successful infections. Here, we report that Fusarium graminearum (Fg), a destructive fungal pathogen of cereal crops, is challenged by host-derived high-iron stress. Fg infection induces host alkalinization, and the pH-dependent transcription factor FgPacC undergoes a proteolytic cleavage into the functional isoform named FgPacC30 under alkaline host environment. Subsequently FgPacC30 binds to a GCCAR(R = A/G)G element at the promoters of the genes involved in iron uptake and inhibits their expression, leading to adaption of Fg to high-iron stress. Mechanistically, FgPacC30 binds to FgGcn5 protein, a catalytic subunit of Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, leading to deregulation of histone acetylation at H3K18 and H2BK11, and repression of iron uptake genes. Moreover, we identified a protein kinase FgHal4, which is highly induced by extracellular high-iron stress and protects FgPacC30 against 26S proteasome-dependent degradation by promoting FgPacC30 phosphorylation at Ser2. Collectively, this study uncovers a novel inhibitory mechanism of the SAGA complex by a transcription factor that enables a fungal pathogen to adapt to dynamic microenvironments during infection.