Cell cycle-regulated histone acetylation required for expression of the yeast HO gene

Transcriptional activation in eukaryotes requires that transcription factors gain access to their target genes in a chromatin environment. The wrapping of DNA around histone proteins to form nucleosomes, as well as the higher-order folding of nucleosomal arrays, produce structures that are inaccessible to the transcription machinery. Naturally, eukaryotes have evolved mechanisms with which to contend with these repressive chromatin structures (Workman and Kingston 1998). In recent years a large number of protein complexes capable of modifying chromatin structure have been identified. These chromatin remodelers fall into two general classes: the ATP-dependent remodelers and the histone acetyltransferases (HATs). The prototype of the ATP-dependent class of remodeling enzymes is the SWI/SNF complex of Saccharomyces cerevisiae, which is required for the transcriptional activation of a subset of genes and for the full functioning of several transcriptional activators (Laurent and Carlson 1992; Laurent et al. 1993; Peterson et al. 1994; Peterson 1996; Burns and Peterson 1997; Ryan et al. 1998). In vitro, SWI/SNF uses the energy of ATP hydrolysis to alter DNA-histone contacts to make nucleosomal DNA more accessible to transcription factors (Cote et al. 1994, 1998; Imbalzano et al. 1994, 1998; Utley et al. 1996) and restriction enzymes (Logie and Peterson 1997). Yeast have also provided the first example of a transcription-related HAT, Gcn5 (Brownell et al. 1996), which is a member of several multisubunit complexes (Grant et al. 1997; Pollard and Peterson 1997; Saleh et al. 1997; Eberharter et al. 1998). GCN5-dependent HAT complexes acetylate nucleosomal histones and can promote transcription from nucleosomal templates in vitro (Steger et al. 1998; Utley et al. 1998; Ikeda et al. 1999). In vivo, GCN5 is required for expression of a set of genes that overlaps with those that require SWI/SNF; in fact, gcn5 swi/snf double mutants exhibit synthetic phenotypes (Pollard and Peterson 1997; Roberts and Winston 1997). Residues in GCN5p that are critical for HAT activity in vitro are also required for Gcn5p’s transcriptional activation function in vivo (Candau et al. 1997; Wang et al. 1998), and GCN5 has been shown to be required for promoter-directed acetylation at the HIS3 gene in vivo (Kuo et al. 1998). Although Gcn5p-containing HAT complexes have been shown to interact with several activator proteins in vitro (Utley et al. 1998), little is known about how these chromain remodelers are targeted to their sites of action in vivo. One of the yeast genes whose expression requires both Gcn5p and SWI/SNF is HO (Pollard and Peterson 1997; Perez-Martin and Johnson 1998), which encodes a site-specific endonuclease that initiates the process of mating-type interconversion. HO expression is tightly regulated by cell type and during the cell cycle, being expressed in only a small window in late G1 and only in haploid mother cells (cells which have budded previously) (for review, see Herskowitz et al. 1992; Nasmyth 1993). This complex pattern of HO expression requires an unusually large upstream regulatory region (∼2000 bp), and a host of positive and negative trans-acting factors, including SWI/SNF, Gcn5p, the sequence-specific activators Swi5p and Swi4p/Swi6p, the Sin3p/Rpd3p deacetylase complex, and the daughter-specific repressor, Ash1p (Breeden and Nasmyth 1987; Sternberg et al. 1987; Herskowitz et al. 1992; Nasmyth 1993; Jansen et al. 1996; Sil and Herskowitz 1996). Swi5p is involved in mother/daughter regulation and binds to two sequence elements located 1300- and 1800-bp upstream from the HO mRNA start site. The SWI4 and SWI6 genes encode two subunits of a transcriptional activator, called SBF, that binds in late G1 to 10 cell cycle boxes located within a 900-bp region upstream of the HO mRNA start site. Binding of SBF in late G1 is not sufficient for activation of HO, as a subsequent step requires phosphorylation by a Cdc28p/Clnp kinase at the G1/S boundary (Start) (Harrington and Andrews 1996; Koch et al. 1996). The inactivation of HO expression after Start requires the activity of an S-phase Cdc28p/Clbp kinase (Amon et al. 1993). In this study, we have examined the histone acetylation pattern of the HO promoter during G1 and early S phase. By taking advantage of mutations in the various regulators of HO, we have determined the activator and remodeler dependence of the changes in histone acetylation. Our results show that there is a cell cycle-regulated wave of GCN5-dependent histone acetylation that is restricted to ∼1 kb of the HO upstream regulatory region encompassing the 10 cell cycle boxes and the TATA element. GCN5-dependent acetylation requires Swi5p and SWI/SNF, but occurs in the absence of Swi4p and precedes transcription. In contrast, inactivation of the Sin3p/Rpd3p deacetylase complex leads to an expanded domain of acetylation as well as high levels of histone acetylation throughout the cell cycle. We propose a sequential order of action for the regulators of HO expression in which the Swi5p activator recruits SWI/SNF whose remodeling activity then targets a Gcn5p-containing HAT complex to the promoter. Acetylation of nucleosomes that encompass the cell cycle boxes may then facilitate binding of the Swi4p/Swi6p complex, which acts very late in the activation cycle. And finally, the Sin3p/Rpd3p deacetylase complex may play a significant role in erasing acetylation events, ensuring that the HO locus has a low level of histone acetylation prior to the beginning of the next cell cycle. These data support the view that chromatin remodeling factors are recruited by site-specific activators, and suggest that two different classes of chromatin remodelers may function at separate and successive steps in gene activation.