The MYC-MAF-SAGA axis drives oncogenic gene expression in multiple myeloma.

The SAGA complex is an evolutionarily conserved histone acetyltransferase complex and transcription coactivator essential for development and disease. Dysregulation of SAGA is implicated in various human diseases, including cancer. In this issue of Genes & Development, Chen et al. (doi:10.1101/gad.351789.124) uncover a critical role for SAGA in multiple myeloma wherein SAGA's ADA2B component is required for the expression of mTORC1 pathway genes and targets of the MYC, E2F, and MAF (musculoaponeurotic fibrosarcoma) transcription factors. SAGA cooperates with MYC and MAF to sustain oncogenic gene expression programs vital for multiple myeloma survival and thus may serve as a therapeutic target for future cancer therapies.

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma.

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of data sets in the Cancer Dependency Map Project revealed that many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and cleavage under targets and release using nuclease assay (CUT&RUN) results identified pathways directly regulated by ADA2B including MTORC1 signaling and oncogenic programs driven by MYC, E2F, and MM-specific MAF. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found that the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

Yeast Mediator facilitates transcription initiation at most promoters via a Tail-independent mechanism.

Mediator (MED) is a conserved factor with important roles in basal and activated transcription. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. Rapid Tail depletion surprisingly reduces transcription from only a small subset of genes. At most of these Tail-dependent genes, in unperturbed conditions, MED is detected at both the UASs and promoters. In contrast, at most Tail-independent genes, we find MED primarily at promoters but not at the UASs. These results suggest that MED Tail and activator-mediated MED recruitment regulates only a small subset of genes. Furthermore, we define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID, and BET factors Bdf1/2. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.

Glucose starvation induces a switch in the histone acetylome for activation of gluconeogenic and fat metabolism genes.

Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters.

Genome-wide, little is understood about how proteins organize at inducible promoters before and after induction and to what extent inducible and constitutive architectures depend on cofactors. We report that sequence-specific transcription factors and their tethered cofactors (e.g., SAGA [Spt-Ada-Gcn5-acetyltransferase], Mediator, TUP, NuA4, SWI/SNF, and RPD3-L) are generally bound to promoters prior to induction ("poised"), rather than recruited upon induction, whereas induction recruits the preinitiation complex (PIC) to DNA. Through depletion and/or deletion experiments, we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 acetylates +1 nucleosomes there. When inducible promoters are poised, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. When induced, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID (transcription factor II-D) to stably associate, rather than creating a completely TFIID-independent PIC, as generally thought. These findings suggest that inducible systems, where present, are integrated with constitutive systems.

The SAGA chromatin-modifying complex: the sum of its parts is greater than the whole.

There are many large protein complexes involved in transcription in a chromatin context. However, recent studies on the SAGA coactivator complex are generating new paradigms for how the components of these complexes function, both independently and in concert. This review highlights the initial discovery of the canonical SAGA complex 23 years ago, our evolving understanding of its modular structure and the relevance of its modular nature for its coactivator function in gene regulation.

Epigenetics and transcription regulation during eukaryotic diversification: the saga of TFIID.

The basal transcription factor TFIID is central for RNA polymerase II-dependent transcription. Human TFIID is endowed with chromatin reader and DNA-binding domains and protein interaction surfaces. Fourteen TFIID TATA-binding protein (TBP)-associated factor (TAF) subunits assemble into the holocomplex, which shares subunits with the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator. Here, we discuss the structural and functional evolution of TFIID and its divergence from SAGA. Our orthologous tree and domain analyses reveal dynamic gains and losses of epigenetic readers, plant-specific functions of TAF1 and TAF4, the HEAT2-like repeat in TAF2, and, importantly, the pre-LECA origin of TFIID and SAGA. TFIID evolution exemplifies the dynamic plasticity in transcription complexes in the eukaryotic lineage.

Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin.

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

Histone Acetylation Inhibits RSC and Stabilizes the +1 Nucleosome.

The +1 nucleosome of yeast genes, within which reside transcription start sites, is characterized by histone acetylation, by the displacement of an H2A-H2B dimer, and by a persistent association with the RSC chromatin-remodeling complex. Here we demonstrate the interrelationship of these characteristics and the conversion of a nucleosome to the +1 state in vitro. Contrary to expectation, acetylation performs an inhibitory role, preventing the removal of a nucleosome by RSC. Inhibition is due to both enhanced RSC-histone interaction and diminished histone-chaperone interaction. Acetylation does not prevent all RSC activity, because stably bound RSC removes an H2A-H2B dimer on a timescale of seconds in an irreversible manner.

Transcript Buffering: A Balancing Act between mRNA Synthesis and mRNA Degradation.

Transcript buffering involves reciprocal adjustments between overall rates in mRNA synthesis and degradation to maintain similar cellular concentrations of mRNAs. This phenomenon was first discovered in yeast and encompasses coordination between the nuclear and cytoplasmic compartments. Transcript buffering was revealed by novel methods for pulse labeling of RNA to determine in vivo synthesis and degradation rates. In this Perspective, we discuss the current knowledge of transcript buffering. Emphasis is placed on the future challenges to determine the nature and directionality of the buffering signals, the generality of transcript buffering beyond yeast, and the molecular mechanisms responsible for this balancing.

TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

KAT tales: Functions of Gcn5 and PCAF lysine acetyltransferases in SAGA and ATAC.

The Allis group identified Gcn5 as the first transcription-related lysine acetyltransferase in 1996, providing a molecular "missing link" between chromatin organization and gene regulation. This review will focus on functions subsequently identified for Gcn5 and the closely related PCAF protein, in the context of two major complexes, SAGA and ATAC, and how the study of these enzymes informs long standing questions regarding the importance of lysine acetylation.

The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22.

The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B-associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus-responsive transcription, where USP22 is required for pre-initiation complex (PIC) stability. Specifically, USP22 maintains long-range enhancer-promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22-independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non-histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription.

De novo variants in ATXN7L3 lead to developmental delay, hypotonia and distinctive facial features.

Deubiquitination is crucial for the proper functioning of numerous biological pathways, such as DNA repair, cell cycle progression, transcription, signal transduction and autophagy. Accordingly, pathogenic variants in deubiquitinating enzymes (DUBs) have been implicated in neurodevelopmental disorders and congenital abnormalities. ATXN7L3 is a component of the DUB module of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex and two other related DUB modules, and it serves as an obligate adaptor protein of three ubiquitin-specific proteases (USP22, USP27X or USP51). Through exome sequencing and by using GeneMatcher, we identified nine individuals with heterozygous variants in ATXN7L3. The core phenotype included global motor and language developmental delay, hypotonia and distinctive facial characteristics, including hypertelorism, epicanthal folds, blepharoptosis, a small nose and mouth, and low-set, posteriorly rotated ears. To assess pathogenicity, we investigated the effects of a recurrent nonsense variant [c.340C>T; p.(Arg114Ter)] in fibroblasts of an affected individual. ATXN7L3 protein levels were reduced, and deubiquitylation was impaired, as indicated by an increase in histone H2Bub1 levels. This is consistent with the previous observation of increased H2Bub1 levels in Atxn7l3-null mouse embryos, which have developmental delay and embryonic lethality. In conclusion, we present clinical information and biochemical characterization supporting ATXN7L3 variants in the pathogenesis of a rare syndromic neurodevelopmental disorder.

SAGA Is a General Cofactor for RNA Polymerase II Transcription.

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.

Enzymatic modules of the SAGA chromatin-modifying complex play distinct roles in Drosophila gene expression and development.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and whether they have any SAGA-independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the histone acetyltransferase (HAT) activity blocks oogenesis, while loss of the H2B deubiquitinase (DUB) activity does not. However, the DUB module regulates a subset of genes in early embryogenesis, and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the HAT module. Our results suggest that the DUB module has functions within SAGA and independent functions.

SAGA Structures Provide Mechanistic Models for Gene Activation.

Two recent reports by Cramer and Ben-Shem and colleagues present high-resolution structures of the yeast SAGA transcription coactivator complex. These are the first to resolve the stoichiometry and structure of the core. The core contains an octamer-like fold, flexibly links the enzymatic modules, and facilitates TBP loading onto TATA promoters.

Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos.

The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process. This article has an associated First Person interview with the first author of the paper.

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.

Complications in Patients with Surgically Altered Gastrointestinal Anatomy Undergoing Endoscopic Retrograde Cholangiopancreatography: 15-Year Experience at a Tertiary Care Center in Latin America.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is a common procedure, but it poses challenges in patients with surgically altered gastrointestinal anatomy (SAGA). Alternative techniques like single-balloon enteroscopy (SBE), double-balloon enteroscopy (DBE), or push enteroscopy (PE) have been used, albeit with potential complications. Limited Latin American data exists on ERCP complications in SAGA patients. Our goal is to describe complications of ERCP in SAGA at a national referral institution. METHODS: Retrospective, single-center cohort study. All SAGA ERCP procedures performed at the Gastrointestinal Endoscopy Department of the National Institute of Medical Sciences and Nutrition Salvador Zubirán from January 2008 to May 2023 were included. Extracted data from records included procedure specifics, endoscope type, success, and complications. Complications were evaluated during procedure and 28-day post-procedure and classified using the AGREE system. RESULTS: A total of 266 procedures in 174 patients were included, 74% were women, and the median age was 44 years. Predominant modified anatomy was Roux-en-Y biliary reconstruction (79%), followed by Whipple procedure (13%) and subtotal gastrectomy with Roux-en-Y reconstruction (6.0%). The main indications were cholangitis with stricture (31%), stricture (19%), and cholangitis (19%). DBE was used in 89%, PE in 7.5%, and SBE in 3.4%. Success rates were 77% endoscopic, 72% technical, and 69% therapeutic; in 30%, the procedure was unsuccessful. Complications happened in 18% of cases, most commonly cholangitis (7.5%), followed by perforation (2.6%) and hemorrhage (1.9%). According to the AGREE classification, 10.9% were grades 1 and 2, 6.4% were grade 3, and 0.4% were grade 4 complications. No significant differences emerged between groups with and without complications. Procedures increased over time, but complications and unsuccessful procedures remained stable. CONCLUSION: ERCP complications align with international data, often not requiring invasive treatment. Enhanced exposure to such cases correlates with fewer complications and failures. Prospective studies are essential to identify complication and failure predictors.