Epigenetic silencing is a term used to describe the heritable transmission of a transcriptionally inactive state. The silent mating type loci HML and HMR and telomeres of the budding yeast Saccharomyces cerevisiae are examples of loci that undergo this type of transcriptional silencing and have served as a paradigm for studying this process.
harbor copies of the mating type information genes, α and a
, respectively. They are involved in mating type interconversion with the actively transcribed MAT
locus. Transcriptional silencing at these loci relies on the existence of cis
-acting DNA regulatory elements, termed silencers (E
), which flank both loci. These elements recruit the DNA binding proteins Rap1, Abf1, and ORC, which then serve to recruit the silent information regulators (Sir) 1, 2, 3, and 4 (11
). The widely accepted view of silencing at these loci (and at telomeres) is that histone tails are deacetylated through the action of Sir2, a NAD-dependent histone deacetylase, creating a binding surface on nucleosomes for the binding of Sir3 and Sir4. Multiple rounds of deacetylation lead to the formation of a Sir2/3/4 polymer that spreads on the nucleosomes of the silent region, altering the chromatin and making it unavailable for transcription. The detailed structure of silent chromatin is not known, and exactly how transcription is prevented is a matter of dispute (6
Sir3 is essential for the establishment and maintenance of the silent state at the HM
loci and telomeres. Genetic, two-hybrid, and biochemical studies have identified interactions of Sir3 with histones H3 and H4, Sir4, Rap1, Abf1, and Sir3 itself (reviewed in references 11
, and 33
). Interestingly, all these interactions are within the C-terminal two-thirds of the Sir3 protein. Nevertheless, expression of a Sir3 construct lacking the N-terminal region (hereafter referred to as the N terminus) is not sufficient to promote silencing at HML
or telomeres in place of the full-length protein (17
). Moreover, coexpression of the same C-terminal construct with the N terminus of Sir3 does lead to silencing (17
). Furthermore, point mutations in the N-terminal region can weaken or abolish silencing (35
). These data suggest that the N terminus of Sir3 plays an important role in transcriptional silencing and that it has the ability to function when separated from the rest of the protein (1
The N-terminal region of Sir3 contains a BAH (bromo-adjacent homology) domain within it. The BAH domain has been identified in several chromatin-associated proteins, including Rsc1 and Rsc2, components of the RSC chromatin remodeling complex in S. cerevisiae
; Dnmt1, the major DNA methyltransferase in mouse and human cells; Orc1, the largest subunit of the origin recognition complex; and others (4
). The majority of what is known about the BAH domain comes from work with Orc1 in S. cerevisiae
. Interestingly, this region of Orc1 is not required for its role in replication but is essential for its role in silencing. Our laboratory demonstrated previously that the Orc1 BAH domain interacts with Sir1 and brings it to the silent locus (38
). Subsequently, the regions essential for this interaction were narrowed down to the Orc1 H domain within its BAH domain and a region in the C-terminal portion of Sir1 termed the Orc1-interacting region (OIR) (2
). This has led to the idea that the BAH domain is a protein-protein interaction module that serves to recruit proteins involved in transcriptional regulation.
The BAH domain of Orc1 (Orc1BAH
) is highly similar in sequence to the BAH domain of Sir3 (50% identity within the first 214 amino acids [aa]), and this region of Orc1 can functionally replace the N terminus of Sir3 in silencing (1
). Even though the Orc1 and Sir3 BAH domains are so similar and Orc1BAH
binds to Sir1, all attempts to detect an interaction between Sir1 and the Sir3 BAH domain (Sir3BAH
) have been unsuccessful. Zhang et al. previously showed that replacing the H domain in Sir3 with the H domain of Orc1 was sufficient to promote an interaction between the C terminus of Sir1 and full-length Sir3 (40
). These data suggest that the H domain, which resides within the BAH domain (see Fig. ), determines the specific protein-protein interaction of the BAH domain. Very recently, the cocrystal structure of Orc1BAH
with the Sir1 OIR has been solved; it provided detailed knowledge of the Orc1BAH
-Sir1 interaction (18
). Sir3 is only 35% identical to
Orc1 within its H domain, and a high degree of variability within BAH domain-containing proteins occurs within this region. This may explain why the Sir1 OIR does not bind to Sir3 and suggests that there may be a difference in the way that the Orc1 and Sir3 BAH domains exert their silencing effects.
FIG. 7. Overall structure of Sir3BAH. (A) Structure of Sir3BAH shown in a ribbon representation. The red dashed line denotes the disordered segment in the structure. The H domain is indicated by a left bracket. This is the domain of Orc1BAH known to bind to the (more ...)
Here, we describe a study of Sir3BAH and Orc1BAH undertaken to characterize their roles in silencing. We show that these domains have the ability to silence the HM loci, at least partially, in the complete absence of the rest of the Sir3 protein. This silencing requires overexpression of SIR1. A gain-of-function point mutation within Sir3BAH allows this module to silence even without extra SIR1. We identify two novel properties of Sir3BAH: first, an ability to bind to oligonucleosomes and DNA and, second, a genetic interaction with the Sir1 N terminus. Finally, we present the crystal structure of Sir3BAH and show that it multimerizes to form a novel helical structure in the crystal, which may reflect its mode of binding to chromatin.