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

Abstract

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.

Keywords: Histone deubiquitination; SAGA; Spt7; Transcription; Yeast.

Fig. 1

Association of Ubp8 with chromatin depends on Spt7. a qPCR analysis of GAL1 gene expression levels in WT and spt7Δ strains. b, c Level of Ubp8-TAP (b) or Sus1-TAP (c) associated with the SAGA-regulated gene GAL1 was monitored using ChIP analysis in WT and spt7Δ strains (b, c) and in the sus1Δ strain (b only, negative control) under inhibitory (glucose) or activation conditions (galactose). d Levels of Ubp8-TAP associated with the PMA1 and YEF3 promoters were monitored using ChIP analysis in WT, sus1Δ and spt7Δ strains. In b–d promoter occupancy level was calculated as the ratio of the IP sample signal to the input signal. A.U., arbitrary units. Error bars denote the SD of at least three independent experiments in each panel. The ratios normalized with respect to an intergenic region from at least three independent experiments are shown. In a–d * indicates one-tailed unpaired Student’s t-test p-value < 0.05. e Inputs (IN) and chromatin-enriched fractions (C) of the Ubp8-TAP strain (WT) and its isogenic mutant spt7Δ, were subjected to western blotting to detect Ubp8 (α-TAP, upper panel). Enrichment of chromatin-associated proteins in the C fraction was determined by detection of total histone H2B (α-H2B, lower panel). Cropped blots are shown for clarity. Full-length blots are presented in Additional file 2: Fig. S3. All samples were run in the same gel

Fig. 2

Participation of Spt7 in Ubp8-dependent H2B deubiquitination. a Whole cell extracts were obtained from WT, sgf73Δ and spt7Δ cells, and levels of H2Bub1 were monitored by western blotting using an anti-α-H2Bub1 antibody. Levels of Pgk1 and total H2B were analyzed as loading controls. Cropped blots are shown for clarity. Full-length blots are presented in Additional file 2: Fig. S4 Rep1/2/3 shown in the figure correspond to three independent replicas. b Quantification of panel a showing the ratio of H2Bub1/H2B of three independent experiments. Error bars represent standard deviation. c Ubp8-TAP was purified via TAP from wild-type (WT, lane 2), sus1Δ (lane 3) and spt7Δ (lane 4) strains. The purified Ubp8-TAPs were then incubated with purified histone H2B (containing HA-ubiquitin modified H2B and unmodified H2B) and in vitro H2Bub1 deubiquitination was assayed. Purified histone H2B alone was incubated with buffer and used as a negative control (lane 1(−)). H2B monoubiquitin levels are indicated at the bottom relative to the level in lane 1(−), which was given an arbitrary value of 1. The values are representative of at least three independent experiments. Cropped blots are shown for clarity. Full-length blots are presented in Additional file 2: Fig. S5

Fig. 3

Spt7 is a key factor for Ubp8 cellular localization. a Localization of Ubp8 tagged with GFP in WT and spt7Δ cells was monitored using fluorescence microscopy. Nuclei were stained with DAPI (DNA). Images were cropped from full sized frames of ~ 250–500 cells and scale bars are set to 5.0 µm. b Boxplot indicating GFP nuclear intensity for Ubp8-GFP (WT) and Ubp8-GFPspt7Δ strains obtained from more than 100 cells

Fig. 4

Ada1 is a key factor in Ubp8 cellular localization. a Localization of Ubp8 tagged with GFP in WT, ada1Δ, spt20Δ and ada1Δspt20Δ cells was monitored using fluorescence microscopy. Nuclei were stained with DAPI (DNA). Images were cropped from full sized frames of ~ 250–500 cells and scale bars are set to 5.0 µm. b Boxplot indicating GFP nuclear intensity for Ubp8-GFP WT, Ubp8-GFPada1Δ, Ubp8-GFPspt20Δ and Ubp8-GFPada1Δspt20Δ strains obtained from more than 100 cells in each case. c Localization of the Sus1 protein tagged with GFP in WT and spt7Δ was monitored using fluorescence microscopy. Nuclei were stained with DAPI (DNA). d Upper panel: levels of Sus1 protein tagged with GFP in WT and spt7Δ mutant cells were analyzed by western blotting of whole-cell extracts using an anti-α-GFP antibody. Levels of Pgk1 protein were monitored as the loading control. Lower panel: serial dilutions of WT and mutant spt7Δ cells, expressing or not expressing Sus1-GFP-tagged proteins as indicated, were grown on YPD plates. Plates were incubated for 48 h at 30 °C

Fig. 5

Intact SAGA–CORE complex influences DUBm functions and localization. a In SAGA–CORE mutants spt7Δ, ada1Δ, spt20Δ SAGA is not assembled (dash lines) and the DUBm is free. This DUBm contains full length Sgf73, including the amino acids that reside inside the CORE. The SAGA–CORE is required for DUBm association with chromatin and its deubiquitinating activity. b Spt7 and Ada1, but not Spt20 are required for normal DUBm nuclear localization. A possible explanation is that assembly of pre-CORE particles facilitates the nuclear transport of the DUBm. SAGA model based on [49]