Active and silenced chromatin domains are often in close juxtaposition to one another, and enhancer and silencer elements operate over large distances to regulate the genes in these domains. The lack of promiscuity in the function of these elements suggests that active mechanisms exist to restrict their activity. Insulators are DNA elements that restrict the effects of long-range regulatory elements. Studies on different insulators from different organisms have identified common themes in their mode of action. Numerous insulators map to promoters of genes or have binding sites for transcription factors and like active chromatin hubs and silenced loci, insulators also cluster in the nucleus. These results bring into focus potential conserved mechanisms by which these elements might function in the nucleus.

Transcriptionally active and inactive domains are frequently found adjacent to one another in the eukaryotic nucleus. To better understand the underlying mechanisms by which domains maintain opposing transcription patterns, we performed a systematic genomewide screen for proteins that may block the spread of silencing in yeast. This analysis identified numerous proteins with efficient silencing blocking activities, and some of these have previously been shown to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID, as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes, as barrier proteins. We demonstrate that histone acetylation and chromatin remodeling occurred at the barrier and correlated with a block to the spread of silencing. Our data suggest that multiple overlapping mechanisms were involved in delimiting silenced and active domains in vivo.The eukaryotic nucleus is organized into active and inactive domains, and the modulation of genes within these domains is mediated by different regulatory elements. Enhancers, promoters, and locus control regions activate genes, whereas silencers repress the transcription of genes. Junctions between the active and inactive gene domains commonly occur along chromosomes (reviewed in reference 54), and specific elements and activities that maintain opposing transcription states in adjacent domains have been previously identified.The budding yeast Saccharomyces cerevisiae also possesses active and inactive chromatin domains, and one of the bestcharacterized chromatin domains is the silenced HMR locus. The genes present at HMR are flanked by silencer elements, and proteins that bind these elements recruit the Sir proteins that mediate silencing. Following recruitment, the Sir proteins are thought to spread along the DNA to form a specialized chromatin state that is inaccessible to various enzymatic probes. The silent domains extend beyond the silencers and up to barrier elements that block the spread (3,13,15,21,40,57).While the left boundary of the silenced chromatin domain at HMR is not well defined, the right boundary has been studied in great detail. The deletion of this barrier element at the native HMR locus leads to an increased spread of silenced chromatin and to concomitant repression of neighboring euchromatic genes (13, 15), while the ectopic insertion of this barrier between a silencer and a promoter blocks the repressive effects of the silencer. A specific tRNA gene acts as a barrier at HMR, and mutations in the promoter of this gene or in the RNA polymerase III transcription factors weaken the barrier activity mediated by this tRNA gene. In addition, mutations in the acetyltransferases Sas2p and Gcn5p advertently affect barrier activity mediated by the tRNA gene.The left boundary of the Sir proteins at HML maps to the upstream activation sequence of YCL069w (3, 37), while the right boundary maps to the promoter of the CHA1 gene (37)-though silencing is believed to terminate at HML-I (4). Howev...

In Saccharomyces cerevisiae, the rapamycin-sensitive TOR signaling pathway plays an essential role in up-regulating translation initiation and cell cycle progression in response to nutrient availability. One of the mechanisms by which TOR regulates cell proliferation is by excluding the GLN3 transcriptional activator from the nucleus and, in consequence, preventing its transcriptional activation therein. We examined the possibility that the TOR cascade could also control the transcriptional activity of Gcn4p, which is known to respond to amino acid availability. The results presented in this paper indicate that GCN4 plays a role in the rapamycinsensitive signaling pathway, regulating the expression of genes involved in the utilization of poor nitrogen sources, a previously unrecognized role for Gcn4p, and that the TOR pathway controls GCN4 activity by regulating the translation of GCN4 mRNA. This constitutes an additional TOR-dependent mechanism which modulates the action of transcriptional activators.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP ؉ -glutamate dehydrogenase (NADP ؉ -GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP ؉ -GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.Two pathways for ammonium assimilation and glutamate biosynthesis have been found in a variety of organisms. The first one, described by Holzer and Schneider in 1957 (12), is mediated by NADP ϩ -glutamate dehydrogenase (NADP ϩ -GDH; EC 1.4.1.4), which catalyzes the reductive amination of 2-oxoglutarate to form glutamate. In an alternative pathway demonstrated by Tempest et al. (25), glutamate is aminated to form glutamine by glutamine synthetase (GS; EC 6.3.1.2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT; EC 1.4.1.13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several microorganisms (2,13,16,23) and in higher plants (18).In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source. Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7). Cloning of the GOGAT structural gene (GLT1) and construction of null GOGAT mutants definitively established that this yeast possesses a single NADH-GOGAT enzyme (4) and that GOGATless mutants (7) which cannot be complemented with GLT1 (unpublished results) are probably impaired in GLT1 regulation. In this paper we report the characterization of strains impaired in either GDH1, GLT1, or both. Our results show that there is a third pathway for glutamate biosynthesis, mediated by an N...

Chd proteins are ATP–dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5′ and 3′ ends of genes, accelerating H3 replacement at the 5′ ends of genes while protecting the 3′ ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3′ ends of genes.