Study of the SAGA deubiquitination module: identification of new modulators and its implication on Spinocerebellar Ataxia Type 7

Regulation of chromatin by epigenetic modifications is a fundamental step during the control of gene expression in eukaryotic cells. The participation of different factors including histone chaperones, chromatin remodeling complexes and histone-modifying complexes regulate chromatin dynamics and ensure the correct metabolism of transcripts that need to be exported to the cytoplasm. In these lines, post-translational modifications including monoubiquitination of histone H2B (H2Bub1) and methylation of histone H3 represent a well-studied histone cross-talk which is required for chromatin integrity and transcription. Additionally, the transition from H2Bub1 to its deubiquitinated form by Ubp8, the DUB enzyme from SAGA (Spt-Ada-Gcn5-acetyltranferase) co-activator complex, is fundamental to obtain a correct gene expression. In this work, we demonstrate that the histone chaperone Asf1 and the Ran-binding protein Mog1, participate in maintaining correct levels of H2Bub1. We show that Mog1 is required for the trimethylation of histone H3 at lysine 4 (H3K4me3), hence, acting as a modulator of histone cross-talk. Mog1 role into gene expression is also demonstrated by its physical and genetically interaction with transcription factors including SAGA and COMPASS complexes. Indeed, we demonstrate that Mog1 interacts genetically with TREX-2 subunits and affects mRNA export. During this work, we have also focused in understanding the molecular mechanisms surrounding Spinocerebellar Ataxia Type 7 (SCA7) which is a rare disease caused by amino acid glutamine (Q) repeats within the DUBm component, ATXN7. Therefore, our interest has been directed towards the study of new mechanisms that trigger SCA7 such as the DUB activity from SAGA complex, protein-protein interaction networks and metabolic profiles.La regulación de la cromatina a través de modificaciones epigenéticas es un paso fundamental durante el control de la expresión génica en células eucariotas. La participación de diferentes factores tales como chaperonas de histonas, complejos de remodelación de la cromatina y complejos modificadores de histonas, regulan la dinámica de la cromatina y garantizan el correcto metabolismo de los transcritos que necesitan ser exportados al citoplasma. De esta forma, las modificaciones postraduccionales que incluyen la monoubicuitinación de la histona H2B (H2Bub1) y la metilación de la histona H3 representan un "cross-talk" de histonas la cual es requerida para la integridad de la cromatina y la transcripción. Además, la transición de H2Bub1 a su forma desubicuitinada por Ubp8, la enzima DUB del complejo co-activador SAGA (Spt-Ada-Gcn5-acetiltranferasa), es necesaria para obtener una expresión génica correcta. En este trabajo, se demuestra que la chaperona de histona Asf1 y la proteína de unión a Ran, Mog1, participan en el mantenimiento de los niveles de H2Bub1. Se demuestra que Mog1 es necesaria para la trimetilación de la histona H3 en la lisina 4 (H3K4me3), actuando como un modulador del "cross-talk" de histonas. El papel de Mog1 en la expresión génica también se demuestra por sus interacciones físicas y genéticas con factores de transcripción, incluyendo los complejos SAGA y COMPASS. Además, demostramos que Mog1 interactúa genéticamente con subunidades de TREX-2 y afecta a la exportación de mRNAs. Durante este trabajo, también nos hemos centrado en la comprensión de los mecanismos moleculares que envuelven a la Ataxia Espinocerebelosa Tipo 7 (SCA7), que es una enfermedad rara causada por una repetición de aminoácidos glutamina (Q) dentro del componente del DUBm, ATXN7. Por lo tanto, nuestro interés se ha dirigido hacia el estudio de nuevos mecanismos que desencadenan SCA7, como la actividad DUB del complejo SAGA, las interacciones proteína-proteína y los perfiles metabólicos.La regulació de la cromatina a través de modificacions epigenètiques és un pas fonamental durant el control de l'expressió gènica en cèl·lules eucariotes. La participació de diferents factors tals com chaperones d'histones, complexos de remodelació de la cromatina i complexos modificadors d'histones, regulen la dinàmica de la cromatina i garanteixen el correcte metabolisme dels transcrits que necessiten ser exportats al citoplasma. D'aquesta forma, les modificacions postraduccionals que inclouen la monoubicuitinació de la histona H2B (H2Bub1) i la metilació de la histona H3 representen un "cross-talk" d'histones la qual és requerida per a la integritat de la cromatina i la transcripció. A més, la transició d'H2Bub1 a la seua forma desubicuitinada per Ubp8, l'enzim DUB del complex co-activador SAGA (Spt-Ada-Gcn5-acetiltranferasa), és necessària per a obtenir una expressió gènica correcta. En aquest treball, es demostra que la chaperona de histona Asf1 i la proteïna d'unió a Ran, Mog1, participen en el manteniment dels nivells d'H2Bub1. Es demostra que Mog1 és necessària per a la trimetilació de la histona H3 en la lisina 4 (H3K4me3), actuant com un modulador del "cross-talk" d'histones. El paper de Mog1 en l'expressió gènica també es demostra per les seues interaccions físiques i genètiques amb factors de transcripció, incloent els complexos SAGA i COMPASS. A més, vam demostrar que Mog1 interactua genèticament amb subunitats de TREX-2 i afecta a l'exportació de mRNA. Durant aquest treball, també ens hem centrat en la comprensió dels mecanismes moleculars que envolten a l'Atàxia Espinocerebelosa Tipus 7 (SCA7), que és una malaltia rara causada per una repetició d'aminoàcids glutamina (Q) dins del component del DUBm, ATXN7. Per tant, el nostre interès s'ha dirigit cap a l'estudi de nous mecanismes que desencadenen SCA7, com l'activitat DUB del complex SAGA, les interacciones proteïna-proteïna i els perfils metabòlics.Oliete Calvo, P. (2017). Study of the SAGA deubiquitination module: identification of new modulators and its implication on Spinocerebellar Ataxia Type 7 [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86155TESI

SAGA-CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity

13 páginas, 5 figuras, 1 tabla. Contiene información suplementaria en: Supplementary information accompanies this paper at https://doi.org/10.1186/s13072-020-00367-3Background: Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results: Here we report that a tetrameric DUBm is assembled independently of the SAGA-CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e., independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild-type cells, suggesting a possible role for SAGA-CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays. Conclusions: Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA-CORE complex and that it includes full-length Sgf73. However, subunits of this SAGA-CORE influence DUBm association with chromatin, its localization and its activity.This study was supported by funds to SR-N from the Spanish MINECO, MICIIN (BFU2014-57636, BFU2015-71978, PGC2018-099872-B-I00) and the Generalitat Valenciana (PROM/2012/061, ACOMP2014/061 and PROMETEO 2016/093). This work was supported by FEDER 2014–2020 and the Ministerio de Economia y Competitividad (MINECO) of Spain. V.G-M was supported by the FPU program from the Ministerio de Educación y Ciencia (AP2009-0917); C.C-N by the Generailtat Valenciana PROMETEO/2016/093; P.O-C by the FPI program from MINECO (BES2012-058587); and M.M-E by the GVA (Val I+D: ACIF/2015/025). The M.I-V lab was co-funded by European Regional Development Funds (ERDF) and the Horizon 2020 Framework Programme of the European Union under the grant agreement 688945 (Euro-BioImaging Prep Phase II).Peer reviewe

Unveiling novel interactions of histone chaperone Asf1 linked to TREX-2 factors Sus1 and Thp1

13 páginas, 7 figuras, 2 yablasAnti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes. These include the assembly and disassembly of histones H3 and H4 from chromatin during replication, transcription, and DNA repair. In addition, Asf1 is required for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109. Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the functional links of Asf1 with other cellular machineries, we employed mass spectrometry coupled to tandem affinity purification (TAP) to investigate novel physical interactions of Asf1. Under different TAP-MS analysis conditions, we describe a new repertoire of Asf1 physical interactions and novel Asf1 post-translational modifications as ubiquitination, methylation and acetylation that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and Nup57, which are all involved in transcription coupled to mRNA export in eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export and GAL1 transcription are not affected in asf1Δ a strong genetic interaction exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1 and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56 acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we provide evidence for a role of Asf1 in histone H2B ubiquitination. This work proposes a functional link between Asf1 and TREX-2 components in histone metabolism at the vicinity of the nuclear pore complex.This work has been supported by MINECO, Spain (BFU2011-23418) and by the GV (PROMETEO/2013/061 Valencian Regional Government) grants to S.R.-N. M.P. is funded by MICINN, Spain (BFU2008-01976), and the GV (ACOMP2011/057 Valencian Regional Government). P.O.-C. and E.G.-O. are holders of a MINECO FPI grant and CIPF PhD grant respectively.Peer reviewe

A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export

17 páginas, 12 figuras.Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis.PO-C, JS-Q and CN-C were supported by the FPI, FPU and PROMETEO (BES-2012058587, FPU15/03862, PROMETEO 2016/093). This study was supported by funds to SR-N from the Spanish MINECO (BFU2011-23418, BFU2014-57636) and the Generalitat Valenciana (PROMETEO 2012/061 and ACOMP2014/061), and to JEP-O (BFU2016-77728-C3-3-P, PROMETEOII 2015/006).Peer reviewe