Differential Cofactor Requirements for Histone Eviction from Two Nucleosomes at the Yeast PHO84 Promoter Are Determined by Intrinsic Nucleosome Stability

Abstract

We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength.

Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast

“…However, a later study showed that the number of nucleosomes at the PHO5 promoter in pho80 gcn5 yeast was unchanged relative to the repressed state in wild type yeast, implying that histone eviction did not occur (68). Similarly, at the yeast PHO84 promoter, activation is accompanied by increased accessibility to the restriction enzyme HpaI and histone eviction in wild type yeast, whereas in gcn5 mutant yeast, a delay in transcriptional induction is accompanied by a corresponding delay in histone eviction, with no alteration in the kinetics of increased HpaI accessibility (69). In another example, activation of the yeast RNR3 promoter in response to DNA damage results in altered MNase cleavage and histone eviction in wild type yeast.…”

The INO80 ATP-Dependent Chromatin Remodeling Complex Is a Nucleosome Spacing Factor

“…This dilemma is evident in the case of activation of the PHO5, PHO8, and PHO84 genes in yeast, where INO80 and SWI/SNF both activate transcription in independent manners (3,53). Our study delineates the intrinsic differences between INO80 and SWI/SNF and points to how they may increase accessibility of chromatinized DNA in distinct ways.…”

“…Both INO80 and SWI/SNF have been shown to act on the PHO5 and PHO8 promoters (3,17,18,53). INO80 mostly recently was found to be involved in DNA repair (33,49).…”

In Vitro Reconstitution of PHO5 Promoter Chromatin Remodeling Points to a Role for Activator-Nucleosome Competition In Vivo

“…Of these, only Swi/Snf (4,16,21,43) and Ino80 (4,59) are involved in opening of the PHO5 promoter, to an extent where their absence causes a measurable delay. The same is true for the histone acetyltransferases Gcn5 (7,24) and Rtt109 (66,67) and the histone chaperones Asf1 (1,32) and Nap1 (15). The degree of cofactor dependency varies with the degree of PHO5 induction (16,32).…”