Abstract
Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NAD-dependent deacetylase Sir2, the scaffolding protein Sir4, and the nucleosome-binding protein Sir3. Transcriptionally active regions present a challenge to SIR complex-mediated de novo heterochromatic silencing due to the presence of antagonistic histone post-translational modifications, including acetylation and methylation. Methylation of histone H3K4 and H3K79 is dependent on monoubiquitination of histone H2B (H2B-Ub). The SIR complex cannot erase H2B-Ub or histone methylation on its own. The deubiquitinase (DUB) Ubp10 is thought to promote heterochromatic silencing by maintaining low H2B-Ub at sub-telomeres. Here, we biochemically characterized the interactions between Ubp10 and the SIR complex machinery. We demonstrate that a direct interaction between Ubp10 and the Sir2/4 sub-complex facilitates Ubp10 recruitment to chromatin via a co-assembly mechanism. Using hydrolyzable H2B-Ub analogs, we show that Ubp10 activity is lower on nucleosomes compared with H2B-Ub in solution. We find that Sir2/4 stimulates Ubp10 DUB activity on nucleosomes, likely through a combination of targeting and allosteric regulation. This coupling mechanism between the silencing machinery and its DUB partner allows erasure of active PTMs and the de novo transition of a transcriptionally active DNA region to a silent chromatin state.
Highlights
Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex
To further understand how Ubp10 is recruited to heterochromatin and contributes to silencing, we began by performing chromatin pulldown assays similar to the SILAC-MS experiments using whole-cell extracts (WCE) in which Ubp10 is TAP-tagged endogenously
We employed multiple biochemical techniques to mechanistically determine how Ubp10 activity is coupled to the assembly of SIR complex heterochromatin
Summary
We previously used stable isotope labeling of amino acids in cell culture and mass spectrometry (SILAC-MS) to identify proteins excluded from heterochromatin [36]. To determine whether Ubp is recruited to heterochromatin directly by the SIR complex, we performed chromatin and heterochromatin pulldowns in the presence of recombinant Ubp purified with a GST tag from Escherichia coli. Sir2/4 does not directly interact with H2B-Ub (Fig. 6F, compare lanes 4 and 7), as this interaction could increase the local substrate concentration of both Ubp and H2B-Ub and enhance DUB activity. From these results, we conclude that the enhancement in Ubp DUB activity on H2B-Ub substrates is a combination of direct recruitment and specific allosteric stimulation by Sir2/4 (Fig. 6G)
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