Structural basis for histone H2B deubiquitination by the SAGA DUB module.
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ABSTRACT:
SUBMITTER: Morgan MT
PROVIDER: S-EPMC4863942 | biostudies-literature | 2016 Feb
REPOSITORIES: biostudies-literature
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Project description:Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by cleaving ubiquitin from specific protein substrates. How their activities are modulated in various cellular contexts remains poorly understood. The yeast deubiquitinase Ubp8 protein is recruited and activated by the SAGA complex and, together with Sgf11, Sus1, and Sgf73, forms a DUB module responsible for deubiquitinating histone H2B during gene expression. Here, we report the crystal structure of the complete SAGA DUB module, which features two functional lobes structurally coupled by Sgf73. In the "assembly lobe," a long Sgf11 N-terminal helix is clamped onto the Ubp8 ZnF-UBP domain by Sus1. In the "catalytic lobe," an Sgf11 C-terminal zinc-finger domain binds to the Ubp8 catalytic domain next to its active site. Our structural and functional analyses reveal a central role of Sgf11 and Sgf73 in activating Ubp8 for deubiquitinating histone H2B and demonstrate how a DUB can be allosterically regulated by its nonsubstrate partners.
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Project description:The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a transcription coactivator that contains a histone H2B deubiquitination activity mediated by its Ubp8 subunit. Full enzymatic activity requires the formation of a quaternary complex, the deubiquitination module (DUBm) of SAGA, which is composed of Ubp8, Sus1, Sgf11, and Sgf73. The crystal structures of the DUBm have shed light on the structure/function relationship of this complex. Specifically, both Sgf11 and Sgf73 contain zinc finger domains (ZnF) that appear essential for the DUBm activity. Whereas Sgf73 N-terminal ZnF is important for DUBm stability, Sgf11 C-terminal ZnF appears to be involved in DUBm function. To further characterize the role of these two zinc fingers, we have solved their structure by NMR. We show that, contrary to the previously reported structures, Sgf73 ZnF adopts a C2H2 coordination with unusual tautomeric forms for the coordinating histidines. We further report that the Sgf11 ZnF, but not the Sgf73 ZnF, binds to nucleosomal DNA with a binding interface composed of arginine residues located within the ZnF ?-helix. Mutational analyses both in vitro and in vivo provide evidence for the functional relevance of our structural observations. The combined interpretation of our results leads to an uncommon ZnF-DNA interaction between the SAGA DUBm and nucleosomes, thus providing further functional insights into SAGA's epigenetic modulation of the chromatin structure.
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Project description:Aneuploidy, the unbalanced segregation of chromosomes during cell division, is recurrent in many tumors and the cause of birth defects and genetic diseases. Centromeric chromatin represents the chromosome attachment site to the mitotic spindle, marked by specialized nucleosomes containing a specific histone variant, CEN-H3/Cse4, in yeast. Mislocalization of Cse4 outside the centromere is deleterious and may cause aberrant chromosome behavior and mitotic loss. For this reason, ubiquitylation by the E3-ubiquitin ligase Psh1 and subsequent proteolysis tightly regulates its restricted localization. Among multiproteic machineries, the SAGA complex is not merely engaged in acetylation but also directly involved in deubiquitylation. In this study, we investigated the role of SAGA-DUB's Ubp8-driven deubiquitylation of the centromeric histone variant Cse4 in budding yeast. We found that Ubp8 works in concert with the E3-ubiquitin ligase Psh1, and that its loss causes defective deubiquitylation and the accumulation of a short ubiquitin oligomer on Cse4. We also show that lack of Ubp8 and defective deubiquitylation increase mitotic instability, cause faster Cse4 proteolysis and induce mislocalization of the centromeric histone outside the centromere. Our data provide evidence for a fundamental role of DUB-Ubp8 in deubiquitylation and the stability of the centromeric histone in budding yeast.
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Project description:The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is a transcriptional co-activator that both acetylates and deubiquitinates histones. The histone acetyltransferase (HAT) subunit, Gcn5, is part of a subcomplex of SAGA called the HAT module. A minimal HAT module complex containing Gcn5 bound to Ada2 and Ada3 is required for full Gcn5 activity on nucleosomes. Deletion studies have suggested that the Ada2 SWIRM domain plays a role in tethering the HAT module to the remainder of SAGA. While recent cryo-EM studies have resolved the structure of the core of the SAGA complex, the HAT module subunits and molecular details of its interactions with the SAGA core could not be resolved. Here we show that the SWIRM domain is required for incorporation of the HAT module into the yeast SAGA complex, but not the ADA complex, a distinct six-protein acetyltransferase complex that includes the SAGA HAT module proteins. In the isolated Gcn5/Ada2/Ada3 HAT module, deletion of the SWIRM domain modestly increased activity but had negligible effect on nucleosome binding. Loss of the HAT module due to deletion of the SWIRM domain decreases the H2B deubiquitinating activity of SAGA, indicating a role for the HAT module in regulating SAGA DUB module activity. A model of the HAT module created with Alphafold Multimer provides insights into the structural basis for our biochemical data, as well as prior deletion studies.
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Project description:DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.
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Project description:Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. Herein, we establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. We further establish that Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. We show that in Drosophila, loss of Sgf11 function causes similar photoreceptor axon-targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, we find that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants.
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