Transcription of the Saccharomyces cerevisiae ARG1 gene is under the control of both positive and negative elements. Activation of the gene in minimal medium is induced by Gcn4. Repression occurs in the presence of arginine and requires the ArgR/Mcm1 complex that binds to two upstream arginine control (ARC) elements. With the recent finding that the E2 ubiquitin conjugase Rad6 modifies histone H2B, we examined the role of Rad6 in the regulation of ARG1 transcription. We find that Rad6 is required for repression of ARG1 in rich medium, with expression increased ϳ10-fold in a rad6 null background. Chromatin immunoprecipitation analysis indicates increased binding of TATA-binding protein in the absence of Rad6. The active-site cysteine of Rad6 is required for repression, implicating ubiquitination in the process. The effects of Rad6 at ARG1 involve two components. In one of these, histone H2B is the likely target for ubiquitination by Rad6, since a strain expressing histone H2B with the principal ubiquitination site converted from lysine to arginine shows a fivefold relief of repression. The second component requires Ubr1 and thus likely the pathway of N-end rule degradation. Through the analysis of promoter constructs with ARC deleted and an arg80 rad6 double mutant, we show that Rad6 repression is mediated through the ArgR/Mcm1 complex. In addition, analysis of an ada2 rad6 deletion strain indicated that the SAGA acetyltransferase complex and Rad6 act in the same pathway to repress ARG1 in rich medium.In response to the role of Rad6/Ubc2 as an E2 ubiquitin conjugase, mutations in its gene affect multiple cellular processes. Rad6 acts with Rad18 in pathways of DNA repair (3-5, 46) and with the E3 ubiquitin ligase Ubr1 in the pathway leading to the degradation of multiubiquitinated protein substrates via the 26S proteasome (22,48,69). Independently of Rad18 and Ubr1, Rad6 is required for transcriptional silencing at telomeres and the HM loci (34). The ability of Rad6 to ubiquitinate histones H2A, H2B, and H3 in vitro (29,30,39) and H2B in vivo (59) has led to the suggestion that its ability to regulate gene expression results from changes in chromatin structure. This idea is supported by findings that disruption of rad6 results in changes in the sites of integration of retrotransposons (47,56) and that a strain with a K123R mutation in the principal ubiquitination site of histone H2B has the same sporulation defect as a strain with rad6 deleted (59).The ARG1 promoter provides a valuable system to study the role of factors involved in the activation and repression of transcription. ARG1 encodes argininosuccinate synthetase, which is required in a pathway that also includes ARG2, ARG5,6, ARG8, ARG3, and ARG4 for the biosynthesis of arginine. Transcription of this group of genes is subject to general amino acid control mediated by the activator protein Gcn4 (14,20,32). ARG1, as well as ARG5,6, ARG8, and ARG3, is also subject to repression by arginine (10,14,16,20,38,50). Arginine repression requires a DNA binding comp...

Transcriptional regulation of the Saccharomyces cerevisiae ARG1 gene is controlled by positive and negative elements. The transactivator Gcn4p is required for activation in minimal medium, while arginine repression requires the ArgR/Mcm1 regulatory complex, which binds to two upstream arginine control elements. We have found that the coordinated regulation of ARG1 requires components of the SAGA chromatin-remodeling complex. Using gcn5 deletion strains and a Gcn5 protein carrying the E173Q mutation in the histone acetyltransferase (HAT) region, we show that the HAT activity of Gcn5p is required for repression of ARG1 in rich medium. Similar increases in expression were seen upon deletion of other SAGA components but not upon deletion of the ADA-specific component, Ahc1p. Chromatin immunoprecipitations using antibodies to acetylated H3 confirmed that a decrease in the level of acetylated histones at the ARG1 promoter correlated with increased ARG1 expression. Up-regulation of ARG1 in the absence of Gcn5p also correlated with increased binding of TATA-binding protein to the promoter. The analysis of promoter deletions showed that Gcn5/Ada repression of ARG1 was mediated through the action of the ArgR/Mcm1 regulatory complex. In addition, studies with minimal medium demonstrated a requirement for the Ada proteins in activation of ARG1. This suggests that SAGA has a dual role at ARG1, acting to repress transcription in rich medium and activate transcription in minimal medium.Transcriptional regulation can occur by alteration of the recruitment or activity of the transcriptional machinery (2,34,70,85). In eucaryotic cells the packaging of DNA into chromatin plays an important role in regulating the accessibility of transcription factors (84). Nucleosomes, the principal packaging element of DNA in the nucleus, may be the primary determinant of accessibility for gene-specific regulatory proteins and the basal transcriptional machinery (26, 83). One mechanism by which chromatin structure can be changed is through the reversible acetylation of lysines within the amino-terminal domains of the histones (34,78,85). Often, enhanced gene expression corresponds with increased acetylation of nucleosomes (33, 70); however, there are cases where histone acetylation appears to be unchanged upon activation (18, 56) and others where increased acetylation correlates with repression (3,10,19,50,54,68,75).The importance of histone acetylation in transcription has been confirmed by the identification of histone acetyltransferase (HAT) activities within coregulatory complexes (79). Saccharomyces cerevisiae GCN5 was originally described as a gene encoding a transcriptional adaptor (28) and was found to be functionally linked to a group of genes whose disruption resulted in reduced toxicity from the overexpression of VP16 (5,12,39,48,58). Subsequently Gcn5p was identified as the catalytic component of two multicomponent HAT complexes, the SAGA and ADA complexes (29, 66), both of which preferentially acetylate nucleosomal histone H3 and to ...

Post-translational modifications of histones play an important role in modulating gene transcription within chromatin. We used the mouse mammary tumor virus (MMTV) promoter, which adopts an ordered nucleosomal structure, to investigate the impact of a specific inhibitor of histone deacetylase, trichostatin A (TSA), on progesterone receptor-activated transcription. TSA induced global histone hyperacetylation, and this effect occurred independently of the presence of hormone. Interestingly, chromatin immunoprecipitation analysis revealed no significant change in the level of acetylated histones associated with the MMTV promoter following high TSA treatment. In human breast cancer cells, in which the MMTV promoter adopts a constitutively "open" chromatin structure, treatment with TSA converted the MMTV promoter into a closed structure. Addition of hormone did not overcome this TSA-induced closure of the promoter chromatin. Furthermore, TSA treatment resulted in the eviction of the transcription factor nuclear factor-1 from the promoter and reduced progesterone receptor-induced transcription. Kinetic experiments revealed that a loss of chromatin-remodeling proteins was coincident with the decrease in MMTV transcriptional activity and the imposition of repressed chromatin architecture at the promoter. These results demonstrate that deacetylase inhibitor treatment at levels that induce global histone acetylation may leave specific regulatory regions relatively unaffected and that this treatment may lead to transcriptional inhibition by mechanisms that modify chromatin-remodeling proteins rather than by influencing histone acetylation of the local promoter chromatin structure.

The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) alpha- and beta-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-beta loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-alpha immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-beta were absent. The cellular localization of PDGF-A within the epithelium and the alpha-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-alpha system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR alpha- and beta-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-alpha mRNAs were seen by reverse transcription-polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-beta were almost not detectable. During the first days of life, PDGF-B and PDGFR-beta genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-beta, and PDGFR-alpha were constant with the only exception of a progressive decrease of PDGFR-alpha in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-beta -/- mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

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