The histone H2A variant H2A.Z (Saccharomyces cerevisiae Htz1) plays roles in transcription, DNA repair, chromosome stability, and limiting telomeric silencing. The Swr1-Complex (SWR-C) inserts Htz1 into chromatin and shares several subunits with the NuA4 histone acetyltransferase. Furthermore, mutants of these two complexes share several phenotypes, suggesting they may work together. Here we show that NuA4 acetylates Htz1 Lys 14 (K14) after the histone is assembled into chromatin by the SWR-C. K14 mutants exhibit specific defects in chromosome transmission without affecting transcription, telomeric silencing, or DNA repair. Functionspecific modifications may help explain how the same component of chromatin can function in diverse pathways. Two classes of enzymes have been implicated in regulating chromatin structure and access to the underlying DNA template. The ATP-dependent chromatin remodeling enzymes use ATP hydrolysis to induce nucleosome mobility or disrupt histone-DNA interactions. The second class of enzymes covalently modify (e.g., lysine acetylation, serine phosphorylation, lysine and arginine methylation, ubiquitylation, or ADP ribosylation) various histones, usually on their N-terminal tails (Strahl and Allis 2000; Jenuwein and Allis 2001). Acetylation is carried out by histone acetyltransferases (HATs), which in Saccharomyces cerevisiae include the Gcn5-containing ADA and SAGA complexes, Hat1, Elongator, NuA3, and NuA4 (for review, see Bottomley 2004). These typically have specificity for distinct lysine residues on certain histone N-terminal tails. The acetylation of lysine residues on the N-terminal tails of histones H3 and H4 neutralizes their positive charge, possibly decreasing their affinity for DNA and facilitating chromatin decompaction and disassembly (Eberharter and Becker 2002). Perhaps more important than simple charge neutralization is the specific pattern of acetylation at individual lysine residues, at least some of which recruit bromodomain-containing proteins (Matangkasombut and Buratowski 2003, and references therein).Further chromatin specialization can be introduced by incorporation of variant histones. The major histones are assembled during DNA replication, but can be replaced by variants at specific locations (for review, see The SWR-C shares several subunits with the NuA4 HAT complex (Kobor et al. 2004;Krogan et al. 2004;Mizuguchi et al. 2004), and expression microarray analysis shows the two complexes have common regulatory targets (Krogan et al. 2004). NuA4 is required for the majority of histone H4 acetylation on Lys 5 (K5), K8, and K12 and some on histone H2A K7 Allard et al. 1999). Htz1, SWR-C, and NuA4 have each been implicated in the maintenance of chromosome stability (Krogan et al. 2004). This function for H2A.Z is conserved in the fission yeast Schizosaccharomyces pombe (Carr et al. 1994) and metazoans (Rangasamy et al. 2004).How NuA4 and SWR-C are functionally connected remains unclear. Htz1 incorporation into chromatin is dependent on SWR-C, but independent of NuA...

The Tup1-Ssn6 general repression complex in Saccharomyces cerevisiae represses a wide variety of regulons. Regulon-specific DNA binding proteins recruit the repression complex, and their synthesis, activity, or localization controls the conditions for repression. Rox1 is the hypoxic regulon-specific protein, and a second DNA binding protein, Mot3, augments repression at tightly controlled genes. We addressed the requirements for Tup1-Ssn6 recruitment to two hypoxic genes, ANB1 and HEM13, by using chromatin immunoprecipitation assays. Either Rox1 or Mot3 could recruit Ssn6, but Tup1 recruitment required Ssn6 and Rox1. We also monitored events during derepression. Rox1 and Mot3 dissociated from DNA quickly, accounting for the rapid accumulation of ANB1 and HEM13 RNAs, suggesting a simple explanation for induction. However, Tup1 remained associated with these genes, suggesting that the localization of Tup1-Ssn6 is not the sole determinant of repression. We could not reproduce the observation that deletion of the Tup1-Ssn6-interacting protein Cti6 was required for induction. Finally, Tup1 is capable of repression through a chromatin-dependent mechanism, the positioning of a nucleosome over the TATA box, or a chromatin-independent mechanism. We found that the rate of derepression was independent of the positioned nucleosome and that the TATA binding protein was excluded from ANB1 even in the absence of the positioned nucleosome. The mediator factor Srb7 has been shown to interact with Tup1 and to play a role in repression at several regulons, but we found that significant levels of repression remained in srb7 mutants even when the chromatin-dependent repression mechanism was eliminated. These findings suggest that the repression of different regulons or genes may invoke different mechanisms.

The hypoxic genes of Saccharomyces cerevisiae are repressed by a complex consisting of the aerobically expressed, sequence-specific DNA-binding protein Rox1 and the Tup1-Ssn6 general repressors. The regulatory region of one well-studied hypoxic gene, ANB1, is comprised of two operators, OpA and OpB, each of which has two strong Rox1 binding sites, yet OpA represses transcription almost 10 times more effectively than OpB. We show here that this difference is due to the presence of a Mot3 binding site in OpA. Mutations in this site reduced OpA repression to OpB levels, and the addition of a Mot3 binding site to OpB enhanced repression. Deletion of the mot3 gene also resulted in reduced repression of ANB1. Repression of two other hypoxic genes in which Mot3 sites were associated with Rox1 sites was reduced in the deletion strain, but other hypoxic genes were unaffected. In addition, the mot3⌬ mutation caused a partial derepression of the Mig1-Tup1-Ssn6-repressed SUC2 gene, but not the ␣2-Mcm1-Tup1-Ssn6-repressed STE2 gene. The Mot3 protein was demonstrated to bind to the ANB1 OpA in vitro. Competition experiments indicated that there was no interaction between Rox1 and Mot3, indicating that Mot3 functions either in Tup1-Ssn6 recruitment or directly in repression. A great deal of evidence has accumulated suggesting that the Tup1-Ssn6 complex represses transcription through both nucleosome positioning and a direct interaction with the basal transcriptional machinery. We demonstrate here that under repressed conditions a nucleosome is positioned over the TATA box in the wild-type ANB1 promoter. This nucleosome was absent in cells carrying a rox1, tup1, or mot3 deletion, all of which cause some degree of derepression. Interestingly, however, this positioned nucleosome was also lost in a cell carrying a deletion of the N-terminal coding region of histone H4, yet ANB1 expression remained fully repressed. A similar deletion in the gene for histone H3, which had no effect on repression, had only a minor effect on the positioned nucleosome. These results indicate that the nucleosome phasing on the ANB1 promoter caused by the Rox1-Mot3-Tup1-Ssn6 complex is either completely redundant with a chromatin-independent repression mechanism or, less likely, plays no role in repression at all.Transcriptional repression in eukaryotic cells often involves the assemblage of large complexes that repress through active mechanisms such as direct interactions with the basal transcriptional machinery and organization of chromatin into repressive structures (16,18,34). The repression of the hypoxic genes in baker's yeast provides an example of such a complex involving the DNA-binding protein Rox1 and the general repression complex 46,47). Our studies have focused on a number of aspects of hypoxic gene regulation, including how differential levels of repression of the hypoxic genes are achieved, how the repression complex forms on the DNA, and how the complex inhibits transcription.The hypoxic genes encode oxygen-related functions in respirati...

Background: Transcription is disruptive to chromatin structure and can expose cryptic promoters. Results: We identify those factors that might regulate cryptic transcription from within inactive and transcribed locations. Conclusion: Nucleosome shielding prevents cryptic transcription, and replication-independent histone replacement is co-operatively mediated by three H3-H4 chaperones. Significance: Understanding how cryptic transcription is regulated and lost histones replaced is of fundamental importance.

Integrating these results, we were able to devise a set of rules that govern the combinatorial interactions between Rox1 and Mot3 to achieve differential repression.