Yeast Sgf73/Ataxin-7 serves to anchor the deubiquitination module into both SAGA and Slik(SALSA) HAT complexes

Abstract

Spinocerebellar ataxia (SCA) is a physically devastating, genetically inherited disorder characterized by abnormal brain function that results in the progressive loss of the ability to coordinate movements. There are many types of SCAs as there are various gene mutations that can cause this disease. SCA types 1-3, 6-10, 12, and 17 result from a trinucleotide repeat expansion in the DNA-coding sequence. Intriguingly, recent work has demonstrated that increased trinucleotde expansions in the SCA7 gene result in defect in the function of the SAGA histone acetyltransferase complex. The SCA7 gene encodes a subunit of the SAGA complex. This subunit is conserved in yeast as the SGF73 gene. We demonstrate that Sgf73 is required to recruit the histone deubiquitination module into both SAGA and the related SliK(SALSA) complex, and to maintain levels of histone ubiquitination, which is necessary for regulation of transcription at a number of genes.

Figure 1

Evolutionary conservation of SGF73 and SGF11. A. Alignment of S. cerevisiae, S. pombe, D. melanogaster, H. sapien orthologues of Ataxin7 reveals the conservation of the Ataxin block, zinc finger(s). Conserved residues are indicated in black and semi-conserved residues in gray. B. Phylogenetic analysis revealed that in addition to SGF73, SGF11 is also a member of the Ataxin-7 family of genes.

Figure 2

Analysis of Ubp8 purifications in the absence of SGF73. A. Mudpit analysis of various Ubp8Tap purifications reveals the effect of the loss of SGF73 and SPT20 versus SGF11 on Ubp8-associated complexes. In the absence of SGF73, Ubp8 only co-purifies with other deubiquitination module components, Sus1 and Sgf11. The numbers represent total peptides obtained for each component. B. Ubp8-associated complexes purified in the absence of SGF73 and SPT20 lose the ability to deubiquitinate histone H2B in vitro.

Figure 3

Deletion of SGF73 results in the loss of the deubiquitination module from the SAGA/SLiK(SALSA) complexes. A. Silver stain of various SAGA/SLiK(SALSA) purifications. Lane 1 marker, Lane 2 Ada2Tap purification, Lane 3 Ada2TAP;sgf73Δ purification, Lane 4 Spt8Tap purification, Lane 5 Spt8TAP;sgf73Δ purification, Lane 6 SLiK(SALSA) purification from an HASpt7-1180TAP strain, where Spt7 lacks the C-terminus required for Spt8 association, Lane 7 HASpt7-1180TAP; sgf73Δ. The subunits that were used for purification are labeled, as well as the band corresponding to Sgf73. B. MudPit analysis reveals that deletion of SGF73 from Spt8, Gcn5 and HA-Spt7-1180TAP (Slik) purifications results in the loss of the deubiquitination module, ubp8, sgf11 and Sus1 from SAGA (numbers are total peptides obtained for each subunit).

Figure 4

Loss of SGF73 leads to the loss of in vitro histone deubiquitination activity, but does not affect HAT activity. A. Quantification of deubiquitination activity associated with various Gcn5-containing HAT complexes in the presence or absence of Sgf73. B. Quantification of the histone acetyltransferase activity associated with various Gcn5-containing HAT complexes in the presence of absence of Sgf73.

Figure 5

Deletion of SGF73 results in a global increase in H2B ubiquitin levels similar to deletion of UBP8, but does not affect H3 acetylation. A. Flag western blot analysis of H2Bub levels: Lane 1 H2BK123R, Lane 2 UBP8Δ, Lane 3 SGF73Δ, Lane 4 SGF73Δ, Lane 5 wildtype. The indicated bands represent FlagH2B, FlagH2Bub and FlagH2BubHA. B. Quantification of H3 lysine 9 Western blot signal demonstrates that deletion of SGF73 does not reduce global histone H3 lysine 9 acetylation when compared with deletion of GCN5.

Figure 6

Similar to a UBP8 deletion, deletion of SGF73 in combination with GCN5 results in a GAL phenotype. Cell growth of the sgf73Δ in combination with gcn5Δ or ubp8Δ. Four-fold serial dilutions were spotted on plates containing rich medium with dextrose (YPD) or with galactose (YP-Gal). Wildtype and mutants were grown at 30°C for ~ 2 days.