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The ATAC and SAGA coactivator complexes assemble co-translationally, but their cellular localization properties and functions are distinct

ABSTRACT: To understand the precise mechanism that guide the formation of multisubunit complexes is of key importance. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the distinct modules of ATAC (ADA Two A Containing) and SAGA (SPT ADA GCN5 Acetyltransferase), two lysine acetyl transferase-containing transcription coactivator complexes, assemble co-translationally in the cytoplasm of mammalian cells. Fully assembled SAGA complex forms in the cytoplasm of mammalian cells and cytoplasmic SAGA acetylates non-histones proteins, before imported in the nucleus. In contrast, ATAC has no cytoplasmic functions as it cannot be detected in the cytoplasm of mammalian cells. However, fully assembled endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic detectability and functions are distinct.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Disease Free

SUBMITTER: Luc Negroni

LAB HEAD: Laszlo Tora

PROVIDER: PXD038695 | Pride | 2023-09-27

REPOSITORIES: Pride

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Project description:SAGA and ATAC are two related transcriptional coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development but the precise role of the two complexes in RNA polymerase II transcription in mammals is less understood. To determine whether SAGA and ATAC have redundant or specific functions dependent on their HAT activities, we compared the effects of HAT inactivation in each complex with that of inactivation of either SAGA or ATAC core subunits in mouse embryonic stem cells (ESCs). We show that core subunits of SAGA or ATAC subunits are required for complex assembly, mouse ESC growth and self-renewal. Additionally, ATAC, but not SAGA subunits are required for ESC viability by regulating the transcription of translation-related genes. Surprisingly, depletion of specific or shared HAT module subunits caused a global decrease in histone H3K9 acetylation, but did not result in significant phenotypic or transcriptional defects. Thus, our results indicate that SAGA and ATAC are differentially required for viability and self-renewal of mouse ESCs by regulating transcription through different pathways, in a HAT-independent manner.

2021-08-11 | PXD027026 | Pride

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

Project description:Co-activator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The core module of SAGA constitutes the structural heart, composed of a histone octamer like structure and two additional proteins. The central histone octamer like core structure consists of six histone fold domain (HFD)-containing proteins, forming three HF pairs (TADA1/TAF12, TAF6L/TAF9/9b, and TAF10/SUPT7L) in the mammalian SAGA complexes, to which adds SUPT3H, which contains an intramolecular HF pair. The yeast homologue of SUPT3H, called Spt3, was shown to interact genetically and biochemically with the TATA binding protein (TBP). Here we investigated the role of SUPT3H in human U2OS and mouse embryonic stem (ES) cells. Using immuno-purification coupled mass spectrometry experiments we show that both human and mouse SAGA can assemble without the double HFD-containing SUPT3H. Nascent RNA-seq experiments indicted that in either U2OS or mouse ESCs lacking SUPT3H only a small subset of genes is deregulated. Consequently, in mouse ESCs SUPT3H is not essential for mouse ESC survival, but is required for ESC growth and self-renewal. In addition, TBP recruitment experiments show no major change in TBP accumulation at gene promoters in the absence of SUPT3H in mouse and human cells. Taken together our data suggest in mammalian cells SUPT3H is not required for the assembly of SAGA, general TBP recruitment to genes and cell survival, but it is important for regulating a limited set of genes.

2022-07-26 | PXD026991 | Pride

Importance of TAF8 regions for TFIID lobe B assembly, or for TAF2 interactions, are required for embryonic stem cell survival

Project description:The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 is forming a histone fold pair in lobe B and connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF core-interacting TAF8 domain, and the proline rich domain of TAF8 interacting with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that TAF8 regions involving in the connection of lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

2022-02-15 | PXD028352 | Pride

SAGA and ATAC histone acetyl transferase complexes regulate distinct sets of genes and ATAC defines a class of p300-independent enhancers

Project description:Metazoan SAGA and ATAC are distinct multi-subunits complexes that share the same catalytic HAT subunit (GCN5 or PCAF). Here we show that these human HAT complexes are targeted to different genomic loci representing functionally distinct regulatory elements both at broadly expressed and tissue specific genes. While SAGA can principally be found at promoters, ATAC is recruited to promoters and enhancers, yet only its enhancer binding is cell-type specific. Furthermore, we show that ATAC functions at a set of enhancers that are not bound by p300, revealing a new class of enhancers not yet identified. These findings demonstrate important functional differences between SAGA and ATAC coactivator complexes at the level of the genome and define a novel role for the ATAC complex in the regulation of a set of enhancers. Examination of SPT20 in two different cell types.

2011-11-05 | E-GEOD-31052 | biostudies-arrayexpress

BS3 intermolecular crosslinking map of SAGA and SLIK (SAGA-like) transcription coactivators

Project description:The SAGA-like complex SLIK is a modified version of the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex. SLIK is formed through C-terminal truncation of the Spt7 SAGA subunit, causing loss of Spt8 that interacts with the TATA-binding protein. SLIK and SAGA are both coactivators of RNA polymerase II transcription in yeast. In addition, both SAGA and SLIK perform chromatin modifications and the two complexes have been speculated to uniquely contribute to transcription regulation. To test the respective contribution of SAGA vs. SLIK in transcription regulation, we assayed the chromatin modifying functions of SAGA vs. SLIK, revealing identical kinetics on minimal substrates in vitro. Furthermore, we determined a low-resolution cryo-EM structure of SLIK, revealing a modular architecture identical to SAGA. Finally, we performed a comprehensive study of DNA-binding properties of both coactivators. Purified SAGA and SLIK both associate with ssDNA and dsDNA with high affinity (KD = 10-17 nM) and the binding is sequence-independent. In conclusion, our study shows that the cleavage of Spt7 and the absence of Spt8 subunit in SLIK neither drive any major conformational differences in its structure compared to SAGA, nor significantly affect HAT, DUB or DNA binding activities in vitro.

2021-04-19 | PXD024640 | Pride

TAF8 MUTATION CAUSES INTELLECUAL DISABILITY, TFIID DISASSEMBLY WITH NO EFFECT ON RNA POLYMERASE II TRANSCRIPTION

Project description:The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID nucleates RNA Polymerase II (Pol II) preinitiation complex formation on gene promoters and thus, is crucial for Pol II transcription. Germline knock out of several mouse TFIID subunits (Tbp, Taf7, Taf8, and Taf10) results in lethality at embryonic day 4.0, demonstrating the fundamental role of holo-TFIID in transcription. We identified a child harboring a splice-site mutation in TAF8, who has intellectual disability, poor growth, progressive spasticity and microcephaly. The c.781-G>A TAF8 mutation in this patient resulted in a frame shift, which affected the final 50 carboxy terminal amino acids of TAF8. We found that the mutant TAF8 protein is unstable and the patient c.781-G>A TAF8 primary fibroblasts did not form canonical TFIID complexes. Astonishingly however, genome-wide RNA pol II occupancy and pre-mRNA transcription on the tested genes was unaffected in the patient’s primary fibroblasts. This study indicates that perturbed TFIID function is less deleterious for transcription in human cells than originally anticipated.

2018-04-16 | PXD008084 | Pride

Genome-wide association of Sus1 with RNAPII and tRNA genes depends on the distinctive SAGA subunits Spt8 and Spt7

Project description:We have analyzed the global effect of the conserved transcription-mRNA export factor Sus1 on transcription and its association with chromatin. We used genomic run-on experiments to show that Sus has a broad impact on the stability of most RNA polymerase II-transcribed genes. Genome association of Sus1 by the chromatin immunoprecipitation technique showed that Sus1 was widely distributed throughout coding regions, tending to accumulate towards the 3â ends of highly transcribed transcription factor IID (TFIID) and Spt-Ada-Gcn5 acetyltransferase (SAGA) dependent genes. This accumulation depends on growth conditions, the transcriptional rate and whether the genes are TFIID- or SAGA-regulated. Validation of Sus1 occupancy data also revealed that Sus1 appears at tRNAs. Concomitantly, deletion of SUS1 leads to tRNA overexpression. In addition, we discovered that distinctive SAGA subunits Spt8 and Spt7 play a key role in Sus1 enrichment at the 3â ends of SAGA-regulated genes upon temperature shift and at tRNAs. Thus, our study identifies the molecular mechanisms by which a SAGA factor is recruited genome-wide, and provides evidence of a more general role for this conserved coactivator in eukaryotic transcription. Genome wide analysis of Sus1 in wild type and Spt7 and Spt8 deletion mutants using ChIP-exo and genomic run-on experiments

2016-05-15 | E-GEOD-65902 | biostudies-arrayexpress

RNA-Seq analysis of ada2M-NM-^T, nrg1M-NM-^T and cir1M-NM-^T and KN99M-NM-1 wildtype cells in capsule inducing and non-inducing conditions

Project description:To contextualize the cryptococcal Spt-Ada-Gcn5 Acetyltransferase (SAGA) member Ada2 in the capsule regulatory network, RNA-Seq was performed on wildtype, ada2M-NM-^T and two other transcription factors, nrg1M-NM-^T and cir1M-NM-^T, which like ada2M-NM-^T are also defective for capsule formation, virulence and filamentation. Mutant and wildtype cells were grown in capsule inducing and non-inducing conditions for 90 minutes and transcriptional profiling was performed by RNA-Seq analysis.

2012-01-01 | E-GEOD-32049 | biostudies-arrayexpress

RNA-Seq analysis of ada2Δ, nrg1Δ and cir1Δ and KN99α wildtype cells in capsule inducing and non-inducing conditions

2012-01-01 | GSE32049 | GEO

The SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma [RNA-Seq]

Project description:Pediatric cancers are frequently driven by fusion or amplification events that result in aberrant transcription factor activity. As transcription factors themselves remain challenging to target, an emerging therapeutic approach for these cancers is to target epigenetic complexes that help maintain oncogenic transcriptional programs. It is therefore critical to identify the complete set of epigenetic modulators maintaining the oncogenic epigenetic landscape of pediatric cancers. Here, we used functional genomic screens to identify epigenetic complexes critical for viability in cell line models of MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We identified multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies in MYCN-amplified neuroblastoma. Integrating ChIP-seq, ATAC-seq, and RNA-seq with targeted protein-degradation and gene editing tools, we characterized the DNA recruitment sites of the SAGA complex in neuroblastoma, and the consequences of SAGA complex lysine acetyltransferase (KAT) activity loss on histone acetylation and gene expression. We demonstrate that loss of SAGA KAT activity suppresses MYC and MYCN gene expression programs and impairs cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable with a KAT2A/KAT2B proteolysis targeting chimera molecule that demonstrated significant activity in vitro and in vivo. 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.

2024-04-19 | GSE211354 | GEO

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