Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly

Abstract

Heterochromatin formation in budding yeast is regulated by the silent information regulator (SIR) complex. The SIR complex comprises the NADdependent 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 PTMs, including acetylation and methylation. Methylation of histone H3K4 and H3K79 are dependent on mono-ubiquitination 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 characterize 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 to 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.

FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics

“…Under the conditions tested, the majority of H2A/H2B-Ub was consumed in less than 5 minutes while almost all of the NCP-Ub remain uncleaved after 60 minutes. Similar behavior was recently observed in experiments using human histones containing a cleavable analogue of a native isopeptide linkage and a GST-Ubp10 fusion (Zukowski et al, 2018). Taken together, our results indicate that Ubp10 discriminates between freestanding histone heterodimers and those in nucleosomes ( Figure 2A) whereas Ubp8/SAGA does not (Morgan et al, 2016).…”

“…Ubp10 contains an unstructured region rich in Asp/Glu that is N-terminal to the catalytic USP domain (residues 362 -733) (Reed et al, 2015) ( Figure 3A). The Nterminal unstructured region contains residues that interact with the Sir3/Sir4 silencing proteins and recruit Ubp10 to subtelomeric regions (Emre et al, 2005;Gardner et al, 2005;Zukowski et al, 2018). However, it is not known how Ubp10 is recruited to ubiquitinated nucleosomes elsewhere in the genome.…”

The Sir4 H‐ BRCT domain interacts with phospho‐proteins to sequester and repress yeast heterochromatin

“…(residues 109-133) that had been recently shown to interact with Sir4 (Zukowski et al, 2018). Based on sequence comparisons with Esc1 and Ty5, we hypothesized that the Ubp10-Sir4 interaction might also be phosphorylation dependent and therefore used the Ubp10 peptide (LSTELSpTEPPSS, residues 117-128) in its Thr123 phosphorylated state (pT123).…”

“…The Sir4 PAD also interacts with the integrase of the Ty5 retrotransposon to integrate Ty5 into heterochromatic regions at telomeres and silent mating-type loci. Finally, an N-terminal region of the Ubp10 deubiquitinase (residues 109-133) has been shown to interact with the Sir4 PAD to remove H2B ubiquitin marks, a prerequisite for heterochromatin formation (Reed et al, 2015;Zukowski et al, 2018). Furthermore, Ty5 binding to Sir4 is mediated by post-translational modifications, such as phosphorylation (Zou et al, 1996;Dai et al, 2007;Brady et al, 2008).…”

“…Furthermore, Ty5 binding to Sir4 is mediated by post-translational modifications, such as phosphorylation (Zou et al, 1996;Dai et al, 2007;Brady et al, 2008). Finally, an N-terminal region of the Ubp10 deubiquitinase (residues 109-133) has been shown to interact with the Sir4 PAD to remove H2B ubiquitin marks, a prerequisite for heterochromatin formation (Reed et al, 2015;Zukowski et al, 2018).…”