Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

To better understand the acute host response to Escherichia coli mastitis, we analyzed gene expression patterns of approximately 23,000 transcripts 4 h after an intramammary infusion of lipopolysaccharide (LPS) in a mouse model. A total of 489 genes were significantly affected, of which 391 were induced and 98 were repressed. Gene ontology analysis demonstrated that most of the induced genes were associated with the innate immune response, apoptosis, and cell proliferation. Substantial induction of the chemokines CXCL1, CXCL2, and S100A8; the acute-phase protein SAA3; and the LPS binding protein CD14 were confirmed by Northern blot analysis. A subsequent time course experiment revealed CXCL1 induction prior to that of CD14 and SAA3. Mammary epithelial cell cultures also showed marked expression of these factors in response to LPS. The expression of immune-related genes in mammary epithelial cells indicates the importance of this cell type in initiating the inflammatory responses. Repressed genes include several carbohydrate and fatty acid metabolic enzymes and potassium transporters, which may contribute to milk composition changes during mastitis. Therefore, the overall transcription profile, in conjunction with gene ontology analysis, provides a detailed picture of the molecular mechanisms underlying the complex biological processes that occur during LPSinduced mastitis.Mastitis is an inflammation of the mammary gland that is usually due to bacterial infection. It is relatively common in lactating women, and incidence rates of 10% to 20% are typically reported, although it has not been extensively studied (5). In contrast, the disease receives considerable attention in the dairy industry because it is the most costly infectious disease of dairy cattle worldwide. In the U.S. dairy industry alone, it causes $2 billion in annual losses due to discarded milk, decreased milk yield and quality, and increased costs for veterinary care and herd management labor (29).Mastitis caused by Escherichia coli is relatively common in high-producing cows, particularly around parturition or during early lactation (8). Though most cows that have E. coli mastitis undergo self-cure, this disease is very acute, and in some cases it can cause severe tissue damage and even death of the animals due to endotoxin (lipopolysaccharide [LPS]) shock. Previous studies of dairy animals have analyzed changes in specific protein production and/or gene expression during experimental challenge or naturally occurring mastitis (3,14,27), but the information provided by these studies is limited to a relatively small number of genes encoding cytokines and chemokines; the expression of other genes involved in mastitis, as well as the signal transduction cascades underlying these changes, is not well defined.Microarray technology has become an important tool to study the complex interactions of host and bacterial pathogens in various infectious-disease settings (6, 9, 26). The major advantage of this technology is the ability to simultaneously monitor d...

The yeast Cyc8 (also known as Ssn6)-Tup1 complex regulates gene expression through a variety of mechanisms, including positioning of nucleosomes over promoters of some target genes to limit accessibility to the transcription machinery. To further define the functions of Cyc8-Tup1 in gene regulation and chromatin remodeling, we performed genome-wide profiling of changes in nucleosome organization and gene expression that occur upon loss of CYC8 or TUP1 and observed extensive nucleosome alterations in both promoters and gene bodies of derepressed genes. Our improved nucleosome profiling and analysis approaches revealed low-occupancy promoter nucleosomes (P nucleosomes) at locations previously defined as nucleosome-free regions. In the absence of CYC8 or TUP1, this P nucleosome is frequently lost, whereas nucleosomes are gained at -1 and +1 positions, accompanying up-regulation of downstream genes. Our analysis of public ChIPseq data revealed that Cyc8 and Tup1 preferentially bind TATA-containing promoters, which are also enriched in genes derepressed upon loss of CYC8 or TUP1. These results suggest that stabilization of the P nucleosome on TATA-containing promoters may be a central feature of the repressive chromatin architecture created by the Cyc8-Tup1 corepressor, and that releasing the P nucleosome contributes to gene activation.

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Here, oligonucleotide array analysis was used to determine gene transcription profiles resulting from these two Ca 2ϩ entry pathways in human cerebrovascular smooth muscle cell cultures. Results were confirmed and expanded using quantitative RT-PCR, Western blot, and immunofluorescence. A distinct, yet overlapping, set of CREregulated genes was induced by VDCC activation using K ϩ membrane depolarization vs. SOCC activation by thapsigargin (TG). Membrane depolarization selectively induced a sustained increase in early growth response-1 (Egr-1) mRNA and protein, which were inhibited by the VDCC blocker nimodipine and the SOCC inhibitor 2-aminoethoxydiphenylborate (2-APB). TG selectively induced a sustained increase in MAPK phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB, but not by nimodipine. The physiological agonist ANG II also stimulated expression of Egr-1 and MKP-1. Coadministration of 2-APB prevented expression of Egr-1 and MKP-1, whereas nimodipine blocked only Egr-1 expression. TG and ANG II induced phosphorylation of ERK, which was sensitive to 2-APB and was selectively required for CRE-binding protein phosphorylation. Our findings thus indicate that Ca 2ϩ entry through VDCCs and store-operated Ca 2ϩ entry can differentially regulate CRE-containing genes in vascular smooth muscle and also imply that agonist-induced signals involved in modulation of gene transcription can be controlled by multiple sources of Ca 2ϩ .