SAGA Structures Provide Mechanistic Models for Gene Activation

Background 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.

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“…A possible explanation is that assembly of pre-CORE particles facilitates the nuclear transport of the DUBm. SAGA model based on [ 49 ] …”

Section: Discussionmentioning

confidence: 99%

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

Nuño-Cabanes

García-Molinero

Martín-Expósito

et al. 2020

Epigenetics & Chromatin

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“…[25] The SAGA complex is a transcriptional coactivator and possesses lysine acetyltransferase activity. [42] It was shown that SAGA is recruited to nucleosomes containing trimethylated H3K4me3 by a tandem Tudor domain with SAGA protein Sgf29. H3K4me3 nucleosomes were generated by F40-SrtA catalyzed SML of H3 from a synthetic H3K4me3 peptide and N-terminal truncated recombinant H3.…”

Section: Applications Of Engineered Sortasesmentioning

confidence: 99%

“…Ligation of the native H3 sequence APATG by F40‐SrtA allows traceless H3 semisynthesis and has already been applied in chromatin biochemistry [25] . The SAGA complex is a transcriptional coactivator and possesses lysine acetyltransferase activity [42] . It was shown that SAGA is recruited to nucleosomes containing trimethylated H3K4me3 by a tandem Tudor domain with SAGA protein Sgf29.…”

Section: Applications Of Engineered Sortasesmentioning

confidence: 99%

Engineered Sortases in Peptide and Protein Chemistry

Freund

Schwarzer

2021

ChemBioChem

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The transpeptidase sortase A of Staphylococcus aureus (Sa-SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in Gram-positive bacteria. This reaction is exploited for sortase-mediated ligation (SML), allowing the site-specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa-SrtA fueled the development of new biocata-lysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa-SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This minireview focuses on recent advances in the field of directed sortase evolution and applications of these tailor-made enzymes in biochemistry.

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“…To count the cells, a segmentation of the DAPI cell nuclei was performed; iii) Statistical analysis. R package software [49] was used to generate the statistical analysis. To determine signi cant values between different experimental groups, the mean data were compared using one-way analysis of variance (ANOVA).…”

Section: In Vitro Deubiquitination Assaymentioning

confidence: 99%

“…A possible explanation is that assembly of pre-CORE particles facilitates the nuclear transport of the DUBm. SAGA model based on [49].…”

Section: In Vitro Deubiquitination Assaymentioning

confidence: 99%

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

Nuño-Cabanes

García-Molinero

Martín-Expósito

et al. 2020

Preprint

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Background: 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 vitroassays. 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.

show abstract

SAGA Structures Provide Mechanistic Models for Gene Activation

Cited by 6 publications

References 10 publications

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

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

Engineered Sortases in Peptide and Protein Chemistry

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

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