Nucleosome Remodeling at the Yeast PHO8 and PHO84 Promoters without the Putatively Essential SWI/SNF Remodeler.

Chromatin remodeling by ATP-dependent remodeling enzymes is crucial for all genomic processes, like transcription or replication. Eukaryotes harbor many remodeler types, and it is unclear why a given chromatin transition requires more or less stringently one or several remodelers. As a classical example, removal of budding yeast PHO8 and PHO84 promoter nucleosomes upon physiological gene induction by phosphate starvation essentially requires the SWI/SNF remodeling complex. This dependency on SWI/SNF may indicate specificity in remodeler recruitment, in recognition of nucleosomes as remodeling substrate or in remodeling outcome. By in vivo chromatin analyses of wild type and mutant yeast under various PHO regulon induction conditions, we found that overexpression of the remodeler-recruiting transactivator Pho4 allowed removal of PHO8 promoter nucleosomes without SWI/SNF. For PHO84 promoter nucleosome removal in the absence of SWI/SNF, an intranucleosomal Pho4 site, which likely altered the remodeling outcome via factor binding competition, was required in addition to such overexpression. Therefore, an essential remodeler requirement under physiological conditions need not reflect substrate specificity, but may reflect specific recruitment and/or remodeling outcomes.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity-dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium-induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening.

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC's presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.

Differential cofactor requirements for histone eviction from two nucleosomes at the yeast PHO84 promoter are determined by intrinsic nucleosome stability.

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.