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.

Sharing the SAGA.

Transcription initiation is a major regulatory step in eukaryotic gene expression. Co-activators establish transcriptionally competent promoter architectures and chromatin signatures to allow the formation of the pre-initiation complex (PIC), comprising RNA polymerase II (Pol II) and general transcription factors (GTFs). Many GTFs and co-activators are multisubunit complexes, in which individual components are organized into functional modules carrying specific activities. Recent advances in affinity purification and mass spectrometry analyses have revealed that these complexes often share functional modules, rather than containing unique components. This observation appears remarkably prevalent for chromatin-modifying and remodeling complexes. Here, we use the modular organization of the evolutionary conserved Spt-Ada-Gcn5 acetyltransferase (SAGA) complex as a paradigm to illustrate how co-activators share and combine a relatively limited set of functional tools.

New insights into the evolutionary conservation of the sole PIKK pseudokinase Tra1/TRRAP.

Phosphorylation by protein kinases is a fundamental mechanism of signal transduction. Many kinase families contain one or several members that, although evolutionarily conserved, lack the residues required for catalytic activity. Studies combining structural, biochemical, and functional approaches revealed that these pseudokinases have crucial roles in vivo and may even represent attractive targets for pharmacological intervention. Pseudokinases mediate signal transduction by a diversity of mechanisms, including allosteric regulation of their active counterparts, assembly of signaling hubs, or modulation of protein localization. One such pseudokinase, named Tra1 in yeast and transformation/transcription domain-associated protein (TRRAP) in mammals, is the only member lacking all catalytic residues within the phosphatidylinositol 3-kinase related kinase (PIKK) family of kinases. PIKKs are related to the PI3K family of lipid kinases, but function as Serine/Threonine protein kinases and have pivotal roles in diverse processes such as DNA damage sensing and repair, metabolic control of cell growth, nonsense-mediated decay, or transcription initiation. Tra1/TRRAP is the largest subunit of two distinct transcriptional co-activator complexes, SAGA and NuA4/TIP60, which it recruits to promoters upon transcription factor binding. Here, we review our current knowledge on the Tra1/TRRAP pseudokinase, focusing on its role as a scaffold for SAGA and NuA4/TIP60 complex assembly and recruitment to chromatin. We further discuss its evolutionary history within the PIKK family and highlight recent findings that reveal the importance of molecular chaperones in pseudokinase folding, function, and conservation.

TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability.

Gene expression regulation is essential for cells to adapt to changes in their environment. Co-activator complexes have well-established roles in transcriptional regulation, but less is known about how they sense and respond to signaling cues. We have previously shown that, in fission yeast, one such co-activator, the SAGA complex, controls gene expression and the switch from proliferation to differentiation in response to nutrient availability. Here, using a combination of genetic, biochemical, and proteomic approaches, we show that SAGA responds to nutrients through the differential phosphorylation of its Taf12 component, downstream of both the TORC1 and TORC2 pathways. Taf12 phosphorylation increases early upon starvation and is controlled by the opposing activities of the PP2A phosphatase, which is activated by TORC1, and the TORC2-activated Gad8AKT kinase. Mutational analyses suggest that Taf12 phosphorylation prevents cells from committing to differentiation until starvation reaches a critical level. Overall, our work reveals that SAGA is a direct target of nutrient-sensing pathways and has uncovered a mechanism by which TORC1 and TORC2 converge to control gene expression and cell fate decisions.

The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

Tra1 has specific regulatory roles, rather than global functions, within the SAGA co-activator complex.

The SAGA complex is a conserved, multifunctional co-activator that has broad roles in eukaryotic transcription. Previous studies suggested that Tra1, the largest SAGA component, is required either for SAGA assembly or for SAGA recruitment by DNA-bound transcriptional activators. In contrast to Saccharomyces cerevisiae and mouse, a tra1Δ mutant is viable in Schizosaccharomyces pombe, allowing us to test these issues in vivo. We find that, in a tra1Δ mutant, SAGA assembles and is recruited to some, but not all, promoters. Consistent with these findings, Tra1 regulates the expression of only a subset of SAGA-dependent genes. We previously reported that the SAGA subunits Gcn5 and Spt8 have opposing regulatory roles during S. pombe sexual differentiation. We show here that, like Gcn5, Tra1 represses this pathway, although by a distinct mechanism. Thus, our study reveals that Tra1 has specific regulatory roles, rather than global functions, within SAGA.

Laboratory horror stories: Poison in the agars.

The fission yeast Schizosaccharomyces pombe is a single-celled eukaryote that can be cultured as a haploid or as a diploid. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our two laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar, from different suppliers, were toxic. Interestingly, the inhibitory effect was attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

Agar lot-specific inhibition in the plating efficiency of yeast spores and cells.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are highly diverged (530 mya), single-celled, model eukaryotic organisms. Scientists employ mating, meiosis, and the plating of ascospores and cells to generate strains with novel genotypes and to discover biological processes. Our three laboratories encountered independently sudden-onset, major impediments to such research. Spore suspensions and vegetative cells no longer plated effectively on minimal media. By systematically analyzing multiple different media components from multiple different suppliers, we identified the source of the problem. Specific lots of agar were toxic. We report that this sporadic toxicity affects independently the agar stocks of multiple vendors, has occurred repeatedly over at least three decades, and extends to species in highly diverged taxa. Interestingly, the inhibitory effects displayed variable penetrance and were attenuated on rich media. Consequently, quality control checks that use only rich media can provide false assurances on the quality of the agar. Lastly, we describe likely sources of the toxicity and we provide specific guidance for quality control measures that should be applied by all vendors as preconditions for their sale of agar.

The structure of the NuA4-Tip60 complex reveals the mechanism and importance of long-range chromatin modification.

Histone acetylation regulates most DNA transactions and is dynamically controlled by highly conserved enzymes. The only essential histone acetyltransferase (HAT) in yeast, Esa1, is part of the 1-MDa NuA4 complex, which plays pivotal roles in both transcription and DNA-damage repair. NuA4 has the unique capacity to acetylate histone targets located several nucleosomes away from its recruitment site. Neither the molecular mechanism of this activity nor its physiological importance are known. Here we report the structure of the Pichia pastoris NuA4 complex, with its core resolved at 3.4-Å resolution. Three subunits, Epl1, Eaf1 and Swc4, intertwine to form a stable platform that coordinates all other modules. The HAT module is firmly anchored into the core while retaining the ability to stretch out over a long distance. We provide structural, biochemical and genetic evidence that an unfolded linker region of the Epl1 subunit is critical for this long-range activity. Specifically, shortening the Epl1 linker causes severe growth defects and reduced H4 acetylation levels over broad chromatin regions in fission yeast. Our work lays the foundations for a mechanistic understanding of NuA4's regulatory role and elucidates how its essential long-range activity is attained.

Quantitative analysis of protein-RNA interactions in fission yeast.

Characterizing the interactions between RNAs and proteins in vivo is key to better understand how organisms regulate gene expression. Here, we describe a robust and quantitative protocol to measure specific RNA-protein interactions in a native context using RNA immunoprecipitation (RIP). We provide a comprehensive experimental framework to detect cotranslational interactions and detail the quantitative analysis of purified RNAs by PCR and high-throughput sequencing. Although we developed the protocol in fission yeast, it can be readily implemented in other yeast species. For complete details on the use and execution of this protocol, please refer to Toullec et al. (2021).

The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells.

Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the phosphoinositide 3 kinase-related kinases (PIKK) family but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the triple T (TTT) complex, is essential for PIKK stabilization and activity. Here, using endogenous auxin-inducible degron alleles, we show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRCs). Transcriptomic analysis revealed that TELO2 contributes to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN), ChIP, and kinetic analyses, we propose a model by which TRRAP directly represses the transcription of IRF9, which encodes a master regulator of interferon-stimulated genes. We have therefore uncovered an unexpected transcriptional repressor role for TRRAP, which we propose contributes to its tumorigenic activity.

What do the structures of GCN5-containing complexes teach us about their function?

Transcription initiation is a major regulatory step in eukaryotic gene expression. It involves the assembly of general transcription factors and RNA polymerase II into a functional pre-initiation complex at core promoters. The degree of chromatin compaction controls the accessibility of the transcription machinery to template DNA. Co-activators have critical roles in this process by actively regulating chromatin accessibility. Many transcriptional coactivators are multisubunit complexes, organized into distinct structural and functional modules and carrying multiple regulatory activities. The first nuclear histone acetyltransferase (HAT) characterized was General Control Non-derepressible 5 (Gcn5). Gcn5 was subsequently identified as a subunit of the HAT module of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, which is an experimental paradigm for multifunctional co-activators. We know today that Gcn5 is the catalytic subunit of multiple distinct co-activator complexes with specific functions. In this review, we summarize recent advances in the structure of Gcn5-containing co-activator complexes, most notably SAGA, and discuss how these new structural insights contribute to better understand their functions.

The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.

The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium.

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.

Transcriptional alterations and chromatin remodeling in polyglutamine diseases.

Transcriptional dysregulation is now thought to be a common feature of polyglutamine disorders, including the spinocerebellar ataxias (SCAs). However, the precise causes of transcriptional alterations and how they relate to the observed phenotype remain elusive. Transcriptional impairment differs in different diseases, possibly reflecting the specific functions of the disease-causing proteins. The SCA7 gene product, ataxin-7, is a subunit of a transcriptional coactivator complex (called STAGA or TFTC) that has histone acetyltransferase activity. Studies on the effect of mutant ataxin-7 on STAGA function suggest that chromatin remodeling and transcriptional alterations are key pathologic events in SCA type 7. These studies could reveal how polyglutamine expansions alter the transcriptional regulation of genes required for neuronal function.

Glutamine-expanded ataxin-7 alters TFTC/STAGA recruitment and chromatin structure leading to photoreceptor dysfunction.

Spinocerebellar ataxia type 7 (SCA7) is one of several inherited neurodegenerative disorders caused by a polyglutamine (polyQ) expansion, but it is the only one in which the retina is affected. Increasing evidence suggests that transcriptional alterations contribute to polyQ pathogenesis, although the mechanism is unclear. We previously demonstrated that the SCA7 gene product, ataxin-7 (ATXN7), is a subunit of the GCN5 histone acetyltransferase-containing coactivator complexes TFTC/STAGA. We show here that TFTC/STAGA complexes purified from SCA7 mice have normal TRRAP, GCN5, TAF12, and SPT3 levels and that their histone or nucleosomal acetylation activities are unaffected. However, rod photoreceptors from SCA7 mouse models showed severe chromatin decondensation. In agreement, polyQ-expanded ataxin-7 induced histone H3 hyperacetylation, resulting from an increased recruitment of TFTC/STAGA to specific promoters. Surprisingly, hyperacetylated genes were transcriptionally down-regulated, and expression analysis revealed that nearly all rod-specific genes were affected, leading to visual impairment in SCA7 mice. In conclusion, we describe here a set of events accounting for SCA7 pathogenesis in the retina, in which polyQ-expanded ATXN7 deregulated TFTC/STAGA recruitment to a subset of genes specifically expressed in rod photoreceptors, leading to chromatin alterations and consequent progressive loss of rod photoreceptor function.

Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast.

Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis. Here we report several principles underlying the assembly and topological organization of the highly conserved SAGA and NuA4 co-activator complexes, which share the Tra1 subunit. We show that Tra1 contributes to the overall integrity of NuA4, whereas, within SAGA, it specifically controls the incorporation of the de-ubiquitination module (DUB), as part of an ordered assembly pathway. Biochemical and functional analyses reveal the mechanism by which Tra1 specifically interacts with either SAGA or NuA4. Finally, we demonstrate that Hsp90 and its cochaperone TTT promote Tra1 de novo incorporation into both complexes, indicating that Tra1, the sole pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the assembly of transcriptional complexes and reveals the contribution of dedicated chaperones to this process.

Both normal and polyglutamine- expanded ataxin-7 are components of TFTC-type GCN5 histone acetyltransferase- containing complexes.

SCA7 (spinocerebellar ataxia type 7) is a neurodegenerative disorder caused by a CAG repeat expansion in the SCA7 gene that leads to elongation of a polyglutamine tract in ataxin-7, a protein of unknown function. Sgf73, a putative yeast orthologue of ataxin-7, has been identified as a new component of the yeast SAGA (Spt/Ada/Gcn5 acetyltransferase) multisubunit complex, a co-activator required for the transcription of a subset of RNA polymerase II-dependent genes. We show here that ataxin-7 is an integral component of mammalian SAGA-like complexes, i.e. the TFTC [TBP (TATA-binding protein)-free TAF (TBP-associated factor) complex] and the STAGA (SPT3/TAF9/GCN5 acetyltransferase) complex. In agreement with this, immunoprecipitation of ataxin-7 retained a histone acetyltransferase activity characteristic of TFTC-like complexes. Moreover, polyglutamine expansion in ataxin-7 did not affect its incorporation into TFTCs/STAGA complexes purified from cells from a SCA7 patient. We demonstrate here that ataxin-7 is the human orthologue of a the yeast SAGA Sgf73 subunit, and is a bona fide subunit of human TFTC-like transcriptional complexes.

Disease progression despite early loss of polyglutamine protein expression in SCA7 mouse model.

Nine neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia type 7 (SCA7) are caused by an expansion of a polyglutamine (polyQ) stretch in the respective proteins. Aggregation of expanded polyQ-containing proteins into the nucleus is a hallmark of these diseases. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of these diseases. Using SCA7 and HD mouse models in which we recently described a retinal phenotype, we investigated whether altered gene expression underlies photoreceptor dysfunction. In both models, rhodopsin promoter activity was early and dramatically repressed, suggesting that downregulation of photoreceptor-specific genes plays a major role in polyQ-induced retinal dysfunction. Because the rhodopsin promoter drives mutant ataxin-7 expression in our SCA7 mice, we also assessed whether downregulation of mutant SCA7 transgene would reverse retinopathy progression and aggregate formation. Although residual expression of mutant ataxin-7 was found negligible from 9 weeks of age, SCA7 transgenic mice showed a progressive decline of photoreceptor activity leading to a complete loss of electroretinographic responses from 1 year of age. At this age, aggregates were cleared in only half of the photoreceptors, indicating that their formation is not fully reversible in this model. We demonstrate here that abolishing full-length mutant ataxin-7 expression did not reverse retinopathy progression in SCA7 mice, raising the possibility that some polyQ-induced pathological events might be irreversible.