Previous studies have revealed many parallels in the cell cycle regulation of the Ace2 and Swi5 transcription factors. Although both proteins begin entry into the nucleus near the start of mitosis, here we show that Ace2 accumulates in the nucleus and binds DNA about 10 min later in the cell cycle than Swi5. We used chimeric fusions to identify the N-terminal region of Ace2 as responsible for the delay, and this same region of Ace2 was required for interaction with Cbk1, a kinase necessary for both transcriptional activation by Ace2 and asymmetric distribution of Ace2. Ace2 and Swi5 also showed differences in prevalence during the cell cycle. Swi5 is apparently degraded soon after nuclear entry, whereas constant Ace2 levels throughout the cell cycle suggest Ace2 is exported from the nucleus. Our work suggests that the precise timing of Ace2 accumulation in the nucleus involves both a nuclear export sequence and a nuclear localization signal, whose activities are regulated by phosphorylation.Ace2 and Swi5 are yeast transcription factors with a number of similarities. Previous work has shown that ACE2 and SWI5 are both transcribed during the G 2 portion of the cell cycle, and the Ace2 and Swi5 proteins accumulate in the nucleus at roughly the same time early in M phase (1-3). Additionally the zinc finger DNA-binding domains of the two proteins are nearly identical, and Ace2 and Swi5 recognize the same DNA sequences in vitro (3, 4). Nonetheless Ace2 and Swi5 activate transcription of different genes in vivo (3, 5). Ace2 activates expression of the CTS1 gene, whereas Swi5 does not. In contrast, Swi5 is required for HO expression, whereas Ace2 is unable to activate HO (3).The mechanisms controlling the cell cycle regulation of Ace2 and Swi5 have been investigated. The G 2 -specific transcription of the ACE2 and SWI5 genes is due to binding sites for the Mcm1-Fkh2-Ndd1 complex in both promoters (6 -10). A nuclear localization signal (NLS) 5 was identified in Swi5, and phosphorylation in the vicinity of the NLS by the Cdc28 cyclindependent kinase prevents nuclear accumulation (1). Swi5 is dephosphorylated at anaphase by the Cdc14 phosphatase (11). Importantly mutation of the three phosphorylated serine residues in Swi5 responsible for down-regulation of the NLS results in constitutive nuclear localization of Swi5. The amino acids in Swi5 that are phosphorylated to regulate nuclear localization are conserved in Ace2 (3), and mutation of these residues results in constitutive nuclear localization (12).Ace2 interacts with the Crm1 nuclear export receptor and contains a nuclear export sequence (NES) (13). A GFP-Ace2 fusion is cytoplasmic, but it is nuclear in the presence of the leptomycin B nuclear export inhibitor (13) or in cells with a mutation in the CRM1 nuclear export protein (14). Importantly a GFP-Swi5 fusion does not respond to leptomycin B. Leucine residues are an essential part of all previously characterized NESs, but mutagenesis of this putative NES region in Ace2 shows that leucines are not required for NES ...

PP2ARts1 controls diverse pathways that influence cell size and may link cell cycle entry to cell growth via the transcription factor Ace2.

We use chromatin immunoprecipitation assays to show that the Gcn5 histone acetyltransferase in SAGA is required for SWI/SNF association with the HO promoter and that binding of SWI/SNF and SAGA are interdependent. Previous results showed that SWI/SNF binding to HO was Gcn5 independent, but that work used a strain with a mutation in the Ash1 daughter-specific repressor of HO expression. Here, we show that Ash1 functions as a repressor that inhibits SWI/SNF binding and that Gcn5 is required to overcome Ash1 repression in mother cells to allow HO transcription. Thus, Gcn5 facilitates SWI/SNF binding by antagonizing Ash1. Similarly, a mutation in SIN3, like an ash1 mutation, allows both HO expression and SWI/SNF binding in the absence of Gcn5. Although Ash1 has recently been identified in a Sin3-Rpd3 complex, our genetic analysis shows that Ash1 and Sin3 have distinct functions in regulating HO. Analysis of mutant strains shows that SWI/SNF binding and HO expression are correlated and regulated by histone acetylation. The defect in HO expression caused by a mutant SWI/SNF with a Swi2(E834K) substitution can be partially suppressed by ash1 or spt3 mutation or by a gain-of-function V71E substitution in the TATA-binding protein (TBP). Spt3 inhibits TBP binding at HO, and genetic analysis suggests that Spt3 and TBP(V71E) act in the same pathway, distinct from that of Ash1. We have detected SWI/SNF binding at the HO TATA region, and our results suggest that SWI/SNF, either directly or indirectly, facilitates TBP binding at HO.The Saccharomyces cerevisiae HO gene encodes an endonuclease that initiates mating-type switching in haploid yeast cells, and the gene is governed by complex transcriptional regulation (for reviews, see references 22, 41, and 62). The gene is expressed only during the late G 1 phase of the cell cycle, and only in mother cells, one of the two progeny after mitotic division. The Ash1 repressor protein is required for this asymmetric expression, as HO is expressed in both mother and daughter cells in an ash1 mutant (14, 76).Chromatin structure plays an important role in transcriptional regulation, including at HO. There are two major classes of chromatin-modifying factors that alleviate the repressive effects of chromatin, the ATP-dependent chromatin-remodeling factors, such as SWI/SNF, and histone acetyltransferases (HATs) that covalently modify the N-terminal tails of histones by acetylation (84). Recent work has shown that transcription factors recruit chromatin-modifying factors to promoters and that at some promoters, the concerted action of chromatinremodeling and HAT complexes is required for gene activation (61). It has been shown for a number of promoters that sequence-specific DNA-binding proteins recruit chromatin remodelers and HATs in a temporal order.Sequential recruitment of transcription factors was first shown at the HO gene (25). HO contains two defined upstream promoter regions, URS1 and URS2, which contain recognition sites for the Swi5 and SBF sequence-specific DNA-binding factors,...

Eukaryotic gene regulation usually involves sequence-specific transcription factors and sequence-nonspecific cofactors. A large effort has been made to understand how these factors affect the average gene expression level among a population. However, little is known about how they regulate gene expression in individual cells. In this work, we address this question by mutating multiple factors in the regulatory pathway of the yeast HO promoter (HOpr) and probing the corresponding promoter activity in single cells using time-lapse fluorescence microscopy. We show that the HOpr fires in an "on/off" fashion in WT cells as well as in different genetic backgrounds. Many chromatin-related cofactors that affect the average level of HO expression do not actually affect the firing amplitude of the HOpr; instead, they affect the firing frequency among individual cell cycles. With certain mutations, the bimodal expression exhibits short-term epigenetic memory across the mitotic boundary. This memory is propagated in "cis" and reflects enhanced activator binding after a previous "on" cycle. We present evidence that the memory results from slow turnover of the histone acetylation marks.

Activation of the Saccharomyces cerevisiae HO promoter is highly regulated, requiring the ordered recruitment of activators and coactivators and allowing production of only a few transcripts in mother cells within a short cell cycle window. We conducted genetic screens to identify the negative regulators of HO expression necessary to limit HO transcription. Known repressors of HO (Ash1 and Rpd3) were identified, as well as several additional chromatin-associated factors including the Hda1 histone deacetylase, the Isw2 chromatin remodeler, and the corepressor Tup1. We also identified clusters of HO promoter mutations that suggested roles for the Dot6/Tod6 (PAC site) and Ume6 repression pathways. We used ChIP assays with synchronized cells to validate the involvement of these factors and map the association of Ash1, Dot6, and Ume6 with the HO promoter to a brief window in the cell cycle between binding of the initial activating transcription factor and initiation of transcription. We found that Ash1 and Ume6 each recruit the Rpd3 histone deacetylase to HO, and their effects are additive. In contrast, Rpd3 was not recruited significantly to the PAC site, suggesting this site has a distinct mechanism for repression. Increases in HO expression and SWI/SNF recruitment were all additive upon loss of Ash1, Ume6, and PAC site factors, indicating the convergence of independent pathways for repression. Our results demonstrate that multiple protein complexes are important for limiting the spread of SWI/SNF-mediated nucleosome eviction across the HO promoter, suggesting that regulation requires a delicate balance of activities that promote and repress transcription.

The Tup1–Cyc8 complex is responsible for repression of a large and diverse collection of genes in Saccharomyces cerevisiae. The predominant view has been that Tup1–Cyc8 functions as a corepressor, actively associating with regulatory proteins and organizing chromatin to block transcription. A new study by Wong and Struhl in this issue of Genes & Development (pp. 2525–2539) challenges nearly 20 years of models by demonstrating that Tup1–Cyc8 functions primarily as a shield to block DNA-binding proteins from recruiting transcriptional coactivators.

The Ace2 transcription factor from budding yeast has both a regulated nuclear localization signal and a regulated nuclear export signal, and Ace2 phosphorylation by the Cbk1 kinase results in Ace2 accumulation in daughter cells but not mothers.

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.