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DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.
Assuntos
Núcleo Celular , DNA , Nanoestruturas , Núcleo Celular/metabolismo , Humanos , DNA/química , DNA/metabolismo , Nanoestruturas/química , RNA Polimerase II/metabolismo , Linhagem Celular Tumoral , Nanotubos/químicaRESUMO
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.
Assuntos
Deficiências do Desenvolvimento , Hipotonia Muscular , Humanos , Hipotonia Muscular/genética , Deficiências do Desenvolvimento/genética , Feminino , Masculino , Pré-Escolar , Criança , Fenótipo , Animais , Adolescente , Sequenciamento do Exoma , Face/anormalidades , Lactente , Fatores de TranscriçãoRESUMO
Transcript buffering entails the reciprocal modulation of mRNA synthesis and degradation rates to maintain stable RNA levels under varying cellular conditions. Current research supports a global, non-sequence-specific connection between mRNA synthesis and degradation, but the underlying mechanisms are still unclear. In this study, we investigated changes in RNA metabolism following acute depletion of TIP60/KAT5, the acetyltransferase subunit of the NuA4 transcriptional coactivator complex, in mouse embryonic stem cells. By combining RNA sequencing of nuclear, cytoplasmic, and newly synthesised transcript fractions with biophysical modelling, we demonstrate that TIP60 predominantly enhances transcription of numerous genes, while a smaller set of genes undergoes TIP60-dependent transcriptional repression. Surprisingly, transcription changes caused by TIP60 depletion were offset by corresponding changes in RNA nuclear export and cytoplasmic stability, indicating gene-specific buffering mechanisms. Similarly, disruption of the unrelated ATAC coactivator complex also resulted in gene-specific transcript buffering. These findings reveal that transcript buffering functions at a gene-specific level and suggest that cells dynamically adjust RNA splicing, export, and degradation in response to individual RNA synthesis alterations, thereby sustaining cellular homeostasis.
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DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.
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Most factors that regulate gene transcription in eukaryotic cells are multimeric, often large, protein complexes. The understanding of the biogenesis pathways of such large and heterogeneous protein assemblies, as well as the dimerization partner choice among transcription factors, is crucial to interpret and control gene expression programs and consequent cell fate decisions. Co-translational assembly (Co-TA) is thought to play key roles in the biogenesis of protein complexes by directing complex formation during protein synthesis. In this review we discuss the principles of Co-TA with a special focus for the assembly of transcription regulatory complexes. We outline the expected molecular advantages of establishing co-translational interactions, pointing at the available, or missing, evidence for each of them. We hypothesize different molecular mechanisms based on Co-TA to explain the allocation "dilemma" of paralog proteins and subunits shared by different transcription complexes. By taking as a paradigm the different assembly pathways employed by three related transcription regulatory complexes (TFIID, SAGA and ATAC), we discuss alternative Co-TA strategies for nuclear multiprotein complexes and the widespread - yet specific - use of Co-TA for the formation of nuclear complexes involved in gene transcription. Ultimately, we outlined a series of open questions which demand well-defined lines of research to investigate the principles of gene regulation that rely on the coordinated assembly of protein complexes.
Assuntos
Regulação Enzimológica da Expressão Gênica , Complexos Multiproteicos , Biossíntese de Proteínas , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas/genética , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , HumanosRESUMO
The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.
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To understand the function of multisubunit complexes, it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here, we demonstrate that the core modules of ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription co-activator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, a SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histone proteins. In contrast, ATAC complex subunits cannot be detected in the cytoplasm of mammalian cells. However, an endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble using co-translational pathways, but their subcellular localization, cytoplasmic abundance, and functions are distinct.
Assuntos
Histona Acetiltransferases , Proteínas de Saccharomyces cerevisiae , Animais , Histona Acetiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Cromatina , Núcleo Celular/metabolismo , Proteínas Fúngicas , Proteínas de Saccharomyces cerevisiae/metabolismo , Mamíferos/metabolismoRESUMO
Core promoters are sites where transcriptional regulatory inputs of a gene are integrated to direct the assembly of the preinitiation complex (PIC) and RNA polymerase II (Pol II) transcription output. Until now, core promoter functions have been investigated by distinct methods, including Pol II transcription initiation site mappings and structural characterization of PICs on distinct promoters. Here, we bring together these previously unconnected observations and hypothesize how, on metazoan TATA promoters, the precisely structured building up of transcription factor (TF) IID-based PICs results in sharp transcription start site (TSS) selection; or, in contrast, how the less strictly controlled positioning of the TATA-less promoter DNA relative to TFIID-core PIC components results in alternative broad TSS selections by Pol II.
Assuntos
Fator de Transcrição TFIID , Transcrição Gênica , Animais , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , TATA Box , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismoRESUMO
To understand the function of multisubunit complexes it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the core 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. In addition, SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histones proteins. In contrast, fully assembled ATAC complex cannot be detected in the cytoplasm of mammalian cells. However, endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related coactivators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic abundance and functions are distinct.
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Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.
Assuntos
Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Humanos , Núcleo Celular/metabolismo , Complexos Multiproteicos/química , Regiões Promotoras Genéticas , Fatores Associados à Proteína de Ligação a TATA/química , Fator de Transcrição TFIID/metabolismoRESUMO
Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1 - the largest protein in the complex - as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.
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The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.
Assuntos
Cromatina , Proteínas Supressoras de Tumor , Acetilação , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Reparo do DNA , Dano ao DNA , NucleossomosRESUMO
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.
Assuntos
RNA Polimerase II , Transativadores , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Histonas , Ubiquitina , Animais , Desenvolvimento Embrionário/genética , Histonas/genética , Histonas/metabolismo , Mamíferos/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Epigenetic histone modifiers are key regulators of cell fate decisions in normal and malignant hematopoiesis. Their enzymatic activities are of particular significance as putative therapeutic targets in leukemia. In contrast, less is known about the contextual role in which those enzymatic activities are exercised and specifically how different macromolecular complexes configure the same enzymatic activity with distinct molecular and cellular consequences. We focus on KAT2A, a lysine acetyltransferase responsible for histone H3 lysine 9 acetylation, which we recently identified as a dependence in acute myeloid leukemia stem cells and that participates in 2 distinct macromolecular complexes: Ada two-A-containing (ATAC) and Spt-Ada-Gcn5-Acetyltransferase (SAGA). Through analysis of human cord blood hematopoietic stem cells and progenitors, and of myeloid leukemia cells, we identify unique respective contributions of the ATAC complex to regulation of biosynthetic activity in undifferentiated self-renewing cells and of the SAGA complex to stabilization or correct progression of cell type-specific programs with putative preservation of cell identity. Cell type and stage-specific dependencies on ATAC and SAGA-regulated programs explain multilevel KAT2A requirements in leukemia and in erythroid lineage specification and development. Importantly, they set a paradigm against which lineage specification and identity can be explored across developmental stem cell systems.
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Histona Acetiltransferases , Leucemia Mieloide Aguda , Acetilação , Hematopoese , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismoRESUMO
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. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.
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Células-Tronco Embrionárias Murinas/metabolismo , Dobramento de Proteína , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Camundongos , Domínios Proteicos , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-two-A-containing) are two related coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development, but the role of these complexes in RNA polymerase II transcription is less understood. To determine whether SAGA and ATAC have redundant or specific functions, we compare 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 are required for complex assembly and mouse ESC growth and self-renewal. Surprisingly, depletion of HAT module subunits causes a global decrease in histone H3K9 acetylation, but does not result in significant phenotypic or transcriptional defects. Thus, our results indicate that SAGA and ATAC are differentially required for self-renewal of mouse ESCs by regulating transcription through different pathways in a HAT-independent manner.
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Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Histona Acetiltransferases/metabolismo , Transativadores/metabolismo , Animais , Histonas/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional/fisiologia , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In somatic cells, RNA polymerase II (Pol II) transcription initiation starts by the binding of the general transcription factor TFIID, containing the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), to core promoters. However, in growing oocytes active Pol II transcription is TFIID/TBP-independent, as during oocyte growth TBP is replaced by its vertebrate-specific paralog TBPL2. TBPL2 does not interact with TAFs, but stably associates with TFIIA. The maternal transcriptome is the population of mRNAs produced and stored in the cytoplasm of growing oocytes. After fertilization, maternal mRNAs are inherited by the zygote from the oocyte. As transcription becomes silent after oocyte growth, these mRNAs are the sole source for active protein translation. They will participate to complete the protein pool required for oocyte terminal differentiation, fertilization and initiation of early development, until reactivation of transcription in the embryo, called zygotic genome activation (ZGA). All these events are controlled by an important reshaping of the maternal transcriptome. This procedure combines cytoplasmic readenylation of stored transcripts, allowing their translation, and different waves of mRNA degradation by deadenylation coupled to decapping, to eliminate transcripts coding for proteins that are no longer required. The reshaping ends after ZGA with an almost total clearance of the maternal transcripts. In the past, the murine maternal transcriptome has received little attention but recent progresses have brought new insights into the regulation of maternal mRNA dynamics in the mouse. This review will address past and recent data on the mechanisms associated with maternal transcriptome dynamic in the mouse.
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Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética , Animais , Feminino , Camundongos , Proteínas Nucleares/metabolismo , Oócitos/metabolismo , Gravidez , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Estabilidade de RNA , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Transcrição Gênica , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismoRESUMO
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.
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Expressão Gênica/genética , Histonas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Animais , Camundongos , Fatores de Transcrição/metabolismo , UbiquitinaçãoRESUMO
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.