The molecular and cellular function of TAF8 in the embryonic brain

© 2016 Dr. Farrah El-SaafinTranscription of all class II genes (all protein coding genes and most ncRNA genes) requires the ordered assembly of a preinitiation complex (PIC). The first step in PIC assembly is the binding of the general transcription factor TFIID, to the promoter of the gene, which is followed by the binding of the other general transcription factors, then RNA pol II. TAF8 is part of at least a subset of TFIID complexes (Guermah et al., 2003). It is essential for the in vitro differentiation of adipocytes, but not myocytes (Guermah et al., 2003). Moreover, it is needed for the survival of the inner cell mass, but not the trophectoderm cells of the blastocyst (Voss et al., 2000). Cell-free studies suggest that TAF8 dramatically affects the overall structure of TFIID (Bieniossek et al., 2013). Embryos lacking Taf8 die at the blastocysts stage of embryonic development (Voss et al., 2000), which precluded analysis of the role of TAF8 at later time points in vivo. It was of interest to know the role of TAF8 at later time points in embryonic development, and I wanted to determine if loss of Taf8 resulted in selective apoptosis in cell types other than the inner cell mass. Prior to this study the molecular role of TAF8 in the structural stability of TFIID and the function of TFIID was highly contentious, and so I wanted to examine the requirements of TAF8 for TFIID function. Therefore the aims of my thesis were to determine the cellular function of TAF8 by characterising the consequences of lack of TAF8 in the developing mouse central nervous system using tissue-specific deletion of the Taf8 gene (Taf8–/loxNesCreT/+), and in doing so, investigate a potential role for TAF8 in the regulation of apoptosis. Another aim was to determine if loss of TAF8 affects the structural stability of TFIID, and general transcription in vivo. The major findings of my thesis were that loss of Taf8 in the embryonic central nervous system resulted in massive apoptosis of the E14.5 cerebral cortex, however brain areas other than the cortex were relatively unaffected. The apoptosis in the cerebral cortex of Taf8–/loxNesCreT/+ embryos was caused by a massive 23-fold elevation in p53 protein, an activation of pro-apoptotic p53 target gene Puma, and subsequent degradation of the pro-survival protein MCL1. Deletion of either p53 or Puma resulted in a complete or partial rescue of the apoptotic Taf8–/loxNesCreT/+ phenotype, respectively. However, the complete rescue of the apoptotic phenotype in the Taf8–/loxNesCreT/+p53-/- brains revealed another TAF8 phenotype, namely a failure of neuronal differentiation. I found that loss of TAF8 in the Taf8–/loxNesCreT/+p53-/- brain did not result in destabilization of the TFIID complex. Nevertheless, transcription was extensively effected in the absence of TAF8, albeit in a very surprising manner. Astonishingly, rather than observing a general failure or reduction in transcription, as one might expect when an essential member of TFIID is missing, a large number of genes were substantially upregulated in the absence of Taf8. Apart from upregulation of bona fide protein coding and ncRNA genes, 5' and 3' regions of genes, intergenic regions and predicted genes were up-regulated in the absence of Taf8, indicating that TAF8 normally represses transcription at least at these regions in the genome or plays a role in determining the boundaries of transcription. Finally, a potential role for TAF8 in the regulation of pre-mRNA splicing was uncovered, as a large number of genes were alternatively spliced, and the “sensor of defective splicing”, Mdm4-short, was activated in the absence of Taf8. In conclusion, I discovered that rather than having an essential role the activation of transcription, TAF8 appears to restrict transcription and facilitates splicing, suggesting that the aberrant transcription and splicing causes apoptotic cell death particularly in the cerebral cortex

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

International audienceUbiquitin (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

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

Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

International audienceAbstract Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3 −/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22 −/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3 −/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3 −/− mESCs, or Atxn7l3 −/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3 −/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation

Co-translational assembly of mammalian nuclear multisubunit complexes

Genes encoding protein complex subunits are often dispersed in the genome of eukaryotes, raising the question how these protein complexes assemble. Here, the authors provide evidence that mammalian nuclear transcription complexes are formed co-translationally to ensure specific and functional interactions

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Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine

Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine