SAGA is a coactivator complex with histone acetyl transferase activity, deubiquitinase activity, and can bind the TATA-binding protein, TBP. Using novel approaches, we recently discovered that SAGA is indispensable for the expression of the majority of genes in yeast. Little is known about the function of SAGA in mammalian cells. This project aims at better understanding the function of the SAGA complex for RNA polymerase II transcription in mammalian cells.
To regulate gene expression, activators bind specific DNA sequences at gene regulatory elements where they recruit coactivator complexes which open the chromatin structure at promoters to facilitate the assembly of the preinitiation complex, composed of general transcription factors and RNAPII. SAGA is a paradigm of a coactivator with several distinct activities including histone modifying activities, binding to TBP and interaction with activator. We recently showed that SAGA acts as a general cofactor required for essentially all RNAPII transcription in budding yeast. Whether this applies in mammals is still unknown, as many co-activators have been generated during metazoan evolution, including the ATAC complex which shares histone acetyltransferase module with SAGA. <br />Of particular interest for the function of SAGA is the involvement of one of its subunits, Ataxin-7 (ATXN7), in SpinoCerebellar Ataxia 7 (SCA7), an inherited neurodegenerative disorder. ATXN7 anchors the DUB module within SAGA and is therefore a main regulator of H2Bub. Intriguingly, polyglutamine (polyQ) expansion of ATXN7, in SCA7 patients, results in a very specific photoreceptor (PR) degeneration, by affecting cell-type specific gene expression. Therefore, our current hypothesis is that ATXN7 within the SAGA complex plays a crucial role in the expression of cell-type specific genes which determine the PR cell identity, and that this role is altered when ATXN7 is mutated in SCA7, ultimately resulting in PR degeneration. <br />To better define the role of SAGA in RNAPII transcription in mammal and to decipher the mechanisms involved in SCA7, we intend to pursue three objectives: <br />1. Role of SAGA and the related ATAC complex in RNAPII transcription in mouse embryonic stem cells <br />2. Role of SAGA deubiquitination activity during embryo development <br />3. Role of SAGA/ATXN7 in photoreceptor cell-type gene expression and its dysfunction in SCA7 retinopathy
We used CRISPR/Cas9 genome editing in mouse embryonic stem cells (ESCs) to inactivate genes encoding subunits of the core SAGA (Supt7l and Supt20) and ATAC (Yeats2 and Zzz3) complexes. Although homozygous null Suptl7-/- and Supt20h-/- ESCs were obtained, we could not isolate any homozygous mutant clone for Yeats2 and Zzz3, suggesting that these genes are essential for ESC survival. We further generated new mutant cells expressing endogenous Yeats2 and Zzz3 fused to the Auxin-Inducible-Degron (AID) sequence and could induce an efficient depletion of these proteins upon auxin treatment. Clonal assays of these mutant cells were performed in different media to measure cell growth and self-renewal of ESC upon depletion of SAGA or ATAC function.
For an accurate measurement of RNAPII transcription, we analyzed RNA synthesis uncoupled from RNA degradation by using metabolic labeling and quantification of newly-transcribed RNA. The different mouse ESC lines were labeled with 4 thiouridine (4-sU) for 10 min. Total RNA were extracted, mixed with drosophila labeled spike-in controls, fragmented by sonication and the labeled newly-synthesized RNA were biotinylated and purified. High-throughput RNA sequencing were performed on total and labeled purified RNA (4sU-seq). Oriented libraries for strand-specific sequencing were generated in collaboration with the IGBMC Microarray and High-Throughput Sequencing Platform and sequencing was performed on an Illumina Hiseq4000 system. Analyses of the 4sU-seq data evidenced an efficient purification of newly synthesized RNA, as revealed by the presence of unprocessed transcripts with a high density of reads in introns and downstream of the polyadenylation signal, as well as unstable transcripts such as upstream antisense RNAs.
1) Functional characterization of SAGA and ATAC mutant cell lines indicated that ATAC is required for ES cell survival, whereas Supt7l or Supt20 appear dispensable for ES cell survival. Both ATAC and SAGA are required for ES cell growth and self-renewal. Quantification of newly synthesized mRNA revealed that SAGA and ATAC predominantly regulate different sets of genes. Surprisingly, depletion of shared or specific HAT module subunits 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 ES cells by regulating transcription through different pathways, but in a HAT-independent manner.
2) To study the role of the SAGA DUB module, we analyzed the phenotypes of Atxn7l3-/- and Usp22-/- embryos. We showed that Atxn7l3 and Usp22 are both required for normal development but Atxn7l3-/- embryos display a much more severe phenotype than Usp22-/- embryos. ChIP-seq experiments revealed a dramatic increase of H2Bub levels in Atxn7l3 null embryos and derived cell lines. Transcriptomic analyses in wild-type and Atxn7l3 mutant ES cells and MEFs evidenced limited changes in gene expression contrasting. These results together indicate that the deubiquitination activity of SAGA does not directly regulate global Pol II transcription.
3) To study the role of SAGA in photoreceptors, we compared the transcriptome and epigenetic marks in wild type and SCA7 mouse retina. Our results indicate a preferential downregulation of highly-expressed photoreceptor identity genes in SCA7 retinopathy. Highly-expressed photoreceptor identity genes have an atypical broad deposition of H3K9 acetylation, correlated with H3K27 hyperacetylation at enhancers and transcription of eRNAs. H3K9 broad acetylation could play a role in the activation of cell-type identity enhancers, and their deregulation would account for the downregulation of highly-expressed genes in SCA7 retinopathy.
SAGA and ATAC complexes are required of mouse ES cell maintenance.
ATAC but not SAGA, is required for ES cell survival.
SAGA and ATAC predominantly regulate different sets of genes in ES cells.
SAGA and ATAC functions in ES cells are essentially non-redundant and HAT-independent.
The DUB activity of SAGA is required for normal embryonic development.
The loss of Atxn7l3 causes a more severe phenotype than the loss of the deubiquitinase Usp22.
Deubiquitination of H2Bub does not directly regulate global Pol II transcription.
Highly-expressed photoreceptor identity genes are downregulated in SCA7 retinopathy.
H3K9 broad acetylation is related to cell-type identity enhancers and eRNA transcription.
Alteration of cell-type identity enhancers accounts for transcriptional dysregulation in SCA7 retina.
1. Helmlinger D., Papai G., DevysD., Tora L. What do the structures of GCN5-containing complexes teach us about their function? Biochim Biophys Acta Gene Regul Mech. 2021 1864(2):194614.
2. Tourigny JP, Schumacher K, Devys D, Zentner GE. Architectural Mediator subunits are differentially essential for global transcription in Saccharomyces cerevisiae. Genetics, Accepted manuscript
3. Wang F, El-Saafin F… Devys D, Vincent SD, Tora L. Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription. Cell Death and Differentiation, Accepted manuscript.
4. Fischer V, … Tora L, Devys D. Two related coactivator complexes SAGA and ATAC control embryonic stem cell selfrenewal through two different acetyltransferase-independent mechanisms. Cell Reports, Under review (http://ssrn.com/abstract=3782006)
5. Niewiadomska-Cimicka A, … Hache A, … Trottier Y. SCA7 mouse cerebellar pathology reveals preferential downregulation of key Purkinje cell-identity genes and shared disease signature with SCA1 and SCA2. Journal of Neuroscience (accepted) (https://www.researchsquare.com/article/rs-27474/v2)
SAGA is a coactivator complex with histone acetyl transferase activity, deubiquitinase activity, and can bind the TATA-binding protein, TBP. Using novel approaches, we recently discovered that SAGA is indispensable for the expression of the majority of genes in yeast. Little is known about the function of SAGA in mammalian cells. This proposal aims at better understanding the function of the SAGA complex for RNA polymerase II transcription in mammalian cells. Specifically, we will determine how the activities of SAGA function during the differentiation of mouse embryonic stem cells (ESCs) into various differentiated cell lineages. For this we will measure the rate of transcription by monitoring nascent mRNA in ESCs that we engineered which lack one or more SAGA subunits. Mouse embryonic development is a highly regulated process, requiring the precise coordination of chromatin modifications and gene expression. To understand the role of SAGA during mouse embryonic development, we will focus on the deubiquitination (DUB) activity of SAGA. Using mouse embryos lacking essential subunits of the DUB module, we will delineate the function of the DUB module during gastrulation and organogenesis. A polyglutamine expansion in ATXN7, which links the DUB module into SAGA, leads to a neurodegenerative disease affecting photoreceptor cells, known as SCA7.We will determine at the molecular level how the mutant ATXN7 can result in the specific photoreceptor degeneration observed in SCA7 patients, using ChIP and RNA-seq on photoreceptors cells from mice harboring a polyglutamine expansion in ATXN7, which mimics the human disease. Altogether, this project will provide a clear understanding of the function of SAGA in mammalian cells, during complex processes including cellular differentiation, and during physiological processes such as gastrulation and organogenesis, and we will also gain a mechanistic understanding of how misregulation of SAGA function can result in human disease.
Monsieur Didier DEVYS (INSTITUT DE GENETIQUE ET DE BIOLOGIE MOLECULAIRE ET CELLULAIRE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IGBMC INSTITUT DE GENETIQUE ET DE BIOLOGIE MOLECULAIRE ET CELLULAIRE
IGBMC INSTITUT DE GENETIQUE ET DE BIOLOGIE MOLECULAIRE ET CELLULAIRE
Help of the ANR 380,658 euros
Beginning and duration of the scientific project: February 2019 - 36 Months