Silent information regulator 3: the Goldilocks of the silencing complex

Abstract

A recent explosion of work surrounds the interactions between Sir3p (Silent Information Regulator 3) and chromatin. We review here the Sir3p functions related to its role in silencing in Saccharomyces cerevisiae. This unusual protein, which is absolutely required for silencing, is distantly related to the highly conserved replication initiator Orc1p, but is itself phylogenetically limited to "post-genome-duplicated" budding yeasts. Several recent studies revise earlier models for Sir3p action. Specifically, the N-terminal bromo-adjacent homology (BAH) domain plays a now well-defined role in silencing, and a picture is emerging in which both termini of Sir3p bind two locations on the nucleosome: (1) the loss of ribosomal DNA silencing (LRS) surface in the nucleosome core, and (2) the N-terminal histone tails for effective silencing at telomeres. We relate Sir3p structure and function, and summarize recent molecular studies of Sir3p/chromatin binding, Sir3p/Dot1p competition, and the possible role of O-Acetyl ADP ribose (O-AADPR) in Sir3p/chromatin binding. We emphasize recent genetic data that provide important new insights and settle controversies created by in vitro work. Finally, we synthesize these ideas to revise the model for how Sir3p mediates silent chromatin formation in yeast, in part through its affinity for the LRS region of the nucleosome, which must be "just right."

Figure 1.

Domain structure of Sir3. (A) The primary domain structure of Sir3p with important domains highlighted in different colors. The first 214 amino acids comprise the BAH domain in blue; the region from amino acids 214 to 532 (tan) is largely unstructured (McBryant et al. 2006); and from amino acids 532 to 978 (green), Sir3p is similar to the CDC6 subfamily of the AAA ATPase domain. CHB1 and CHB2 (blue) indicate the C-terminal histone-binding (CHB) activity associated with this portion of Sir3p. The BAH domain also contains histone-binding activity. (B) A cartoon version of the tertiary structure of Sir3 based on the crystal structures of the BAH domain 2FL7 (Hou et al. 2006) and the Archaeal cdc6 protein ortholog 1FNN (chain A) (Liu et al. 2000). The BAH domain is colored in blue, and the C-terminal domain is colored in teal. The red oval placed between domains I and II of the C-terminal region of Sir3 indicates a possible nucleotide moiety such as O-AADPR that could bind to Sir3p.

Figure 2.

Critical binding surface of the BAH domain revealed by multiple genetic screens. (A) Multiple sequence alignment of SIR3 BAH domain from S. cerevisiae, consensus sequence for SIR3 BAH domain from post-genome duplication yeast strains, and BAH domain from ORC1. The side chains identified in the eso, DNG, and lfm screens are highlighted in yellow-green, medium green, and light green, respectively; side chains from the slr screen are highlighted in red; residues overlapping between the slr screens and other loss-of-function screens are highlighted in brown; residues overlapping among the loss-of-function screens DNG, eso, and lfm are highlighted in dark green. Mutations in the slr screen that introduce an “Orc-like” residue are highlighted in purple. Conserved residues are highlighted in blue, and semiconserved residues are highlighted in gray. (B) Surface representation of the crystal structure 2FL7 (Hou et al. 2006). The color coding is similar to A, but simplified; the DNG, eso, and lfm screens are highlighted in green; the slr screen is highlighted in red; and the overlap between the two groups is highlighted in brown. The structure is rotated 180° around the X-axis to demonstrate that all of the side chains identified by the various screens are found on one face of the BAH domain.

Figure 3.

Competing actions for silencing followed by reinforcements. This figure is largely based on Altaf et al. (2007) and Buchberger et al. (2008). There are silencing-promoting actions such as Sir2p deacetylating the histone tails, which creates a high-affinity site for the SIR complex. Several actions inhibit silencing: One is the inherent instability of the SIR complex, and a second is the competition between Sir2p and Sas2p for the acetylation state of histone H4 K16. HTZ1 deposition also contributes to the destabilization of silencing (Venkatasubrahmanyam et al. 2007). Acetylation of H4 K16 leads to decreased affinity of the SIR complex for the nucleosomes and especially for that of Sir3 and the nucleosome; this loss of affinity is reinforced by the competition between Dot1p and Sir3p for the methylation status of histone H3 K79. Sir3p binds the LRS surface of the nucleosome, which encompasses H3 K79 and occludes its methylation by Dot1p; conversely, when H3 K79 is methylated, Sir3p binding is blocked. The orientation of Sir3p is meant to suggest that the N-terminal BAH and the C terminus of Sir3p may bind to both the LRS domain and the histone N-terminal tails, and that this dual binding may in fact be crucial to compaction of chromatin and silencing. Additionally, the Sir3 C terminus is shown to be most sensitive to the actelyation of H4 K16, which inhibits Sir3 “lockdown” onto the chromatin (Johnson et al. 2009), and hence spreading of the silent chromatin. Sir4p is depicted as binding to linker DNA as suggested in Martino et al. (2009), but this aspect of the model should be considered preliminary. The stoichiometry of the SIR complex is also preliminary, and is based primarily on Kimura and Horikoshi (2004) and Johnson et al. (2009).