Dissecting the cis and trans elements that regulate adjacent-gene coregulation in Saccharomyces cerevisiae.

The relative positions that genes occupy on their respective chromosomes can play a critical role in determining how they are regulated at the transcriptional level. For example, a significant fraction of the genes from a variety of coregulated gene sets, including the ribosomal protein (RP) and the rRNA and ribosome biogenesis (RRB) regulons, exist as immediate, adjacent gene pairs. These gene pairs occur in all possible divergent, tandem, and convergent orientations. Adjacent-gene pairing in these regulons is associated with a tighter transcriptional coregulation than is observed for nonpaired genes of the same regulons. In order to define the cis and trans factors that regulate adjacent-gene coregulation (AGC), we conducted a mutational analysis of the convergently oriented RRB gene pair MPP10-YJR003C. We observed that coupled corepression of the gene pair under heat shock was abrogated when the two genes were separated by an actively expressed RNA polymerase (Pol) II transcription unit (the LEU2 gene) but not when the inserted LEU2 gene was repressed. In contrast, the insertion of an RNA Pol III-transcribed tRNA (Thr) gene did not disrupt the coregulated repression of MPP10 and YJR003C. A targeted screen of mutants defective in regulating chromosome architecture revealed that the Spt20, Snf2, and Chd1 proteins were required for coupling the repression of YJR003C to that of MPP10. Nucleosome occupancy assays performed across the MPP10 and YJR003C promoter regions revealed that the mechanism of corepression of the gene pair was not related to the repositioning of nucleosomes across the respective gene promoters.

A new system for naming ribosomal proteins.

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

The adjacent positioning of co-regulated gene pairs is widely conserved across eukaryotes.

BACKGROUND: Coordinated cell growth and development requires that cells regulate the expression of large sets of genes in an appropriate manner, and one of the most complex and metabolically demanding pathways that cells must manage is that of ribosome biogenesis. Ribosome biosynthesis depends upon the activity of hundreds of gene products, and it is subject to extensive regulation in response to changing cellular conditions. We previously described an unusual property of the genes that are involved in ribosome biogenesis in yeast; a significant fraction of the genes exist on the chromosomes as immediately adjacent gene pairs. The incidence of gene pairing can be as high as 24% in some species, and the gene pairs are found in all of the possible tandem, divergent, and convergent orientations. RESULTS: We investigated co-regulated gene sets in S. cerevisiae beyond those related to ribosome biogenesis, and found that a number of these regulons, including those involved in DNA metabolism, heat shock, and the response to cellular stressors were also significantly enriched for adjacent gene pairs. We found that as a whole, adjacent gene pairs were more tightly co-regulated than unpaired genes, and that the specific gene pairing relationships that were most widely conserved across divergent fungal lineages were correlated with those genes that exhibited the highest levels of transcription. Finally, we investigated the gene positions of ribosome related genes across a widely divergent set of eukaryotes, and found a significant level of adjacent gene pairing well beyond yeast species. CONCLUSION: While it has long been understood that there are connections between genomic organization and transcriptional regulation, this study reveals that the strategy of organizing genes from related, co-regulated pathways into pairs of immediately adjacent genes is widespread, evolutionarily conserved, and functionally significant.

Adjacent gene pairing plays a role in the coordinated expression of ribosome biogenesis genes MPP10 and YJR003C in Saccharomyces cerevisiae.

The rRNA and ribosome biogenesis (RRB) regulon from Saccharomyces cerevisiae contains some 200 genes, the expression of which is tightly regulated under changing cellular conditions. RRB gene promoters are enriched for the RRPE and PAC consensus motifs, and a significant fraction of RRB genes are found as adjacent gene pairs. A genetic analysis of the MPP10 promoter revealed that both the RRPE and PAC motifs are important for coordinated expression of MPP10 following heat shock, osmotic stress, and glucose replenishment. The association of the RRPE binding factor Stb3 with the MPP10 promoter was found to increase after glucose replenishment and to decrease following heat shock. Similarly, bulk histone H3 clearing and histone H4K12 acetylation levels at the MPP10 promoter were found to increase or decrease following glucose replenishment or heat shock, respectively. Interestingly, substitutions in the PAC and RRPE sequences at the MPP10 promoter were also found to impact the regulated expression of the adjacent RRB gene YJR003, whose promoter lies in the opposite orientation and some 3.8 kb away. Furthermore, the regulated expression of YJR003C could be disrupted by inserting a reporter cassette that increased its distance from MPP10. Given that a high incidence of gene pairing was also found within the ribosomal protein (RP) and RRB regulons across different yeast species, our results indicate that immediately adjacent positioning of genes can be functionally significant for their coregulated expression.

The budding yeast rRNA and ribosome biosynthesis (RRB) regulon contains over 200 genes.

The ribosome biogenesis pathway constitutes one of the major metabolic obligations for a dividing yeast cell and it depends upon the activity of hundreds of gene products to produce the necessary rRNA and ribosomal protein components. Previously, we reported that a set of 65 S. cerevisiae genes that function in the rRNA biosynthesis pathway are transcriptionally co-regulated as cells pass through a variety of physiological transitions. By analysing multiple microarray-based transcriptional datasets, we have extended that study and now suggest that the ribosomal and rRNA biosynthesis regulon contains over 200 genes. This regulon is distinct from the set of ribosomal protein genes, and the promoters of the expanded RRB gene set are highly enriched for the PAC and RRPE motifs. Since a similar pattern of organization and gene regulation can be recognized in C. albicans, the RRB regulon appears to be a conserved, extensive, and metabolically important group of genes.

The yeast rRNA biosynthesis factor Ebp2p is also required for efficient nuclear division.

Molecular genetic analysis of the yeast Ebp2 protein has revealed that it is an essential, nucleolar protein that functions in the rRNA biosynthesis pathway. Temperature-sensitive ebp2-1 mutants are defective in the processing of the 27 SA precursor rRNA, and the point substitutions that disrupt this activity cluster towards the central, more highly conserved region of the Ebp2 protein. We report here that other ebp2 mutants exhibit deficiencies associated with defects in chromosome segregation. Yeast cells bearing a 50 amino acid C-terminal truncation allele (ebp2 delta C50) display a slow-growth phenotype and exhibit an increased percentage of cells with the nucleus positioned at the bud neck. The ebp2-1 and ebp2 delta C50 alleles genetically complement each other, and ebp2 delta C50 mutants exhibit nuclear division defects that are distinct from the rRNA biosynthesis-related phenotypes of ebp2-1 mutants. Cytological and FACS analysis of the ebp2 delta C50 deletion mutants indicate that the chromosome segregation related activities of the Ebp2 protein are monitored by Mad2p, a mitotic checkpoint protein. The finding that yeast Ebp2p functions in nuclear division is consistent with the growing body of evidence that supports the role that human EBP2 plays in chromosome segregation.

Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC.

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

Two mcm3 mutations affect different steps in the initiation of DNA replication.

Mcm3 is a subunit of the hexameric MCM2-7 complex required for the initiation and elongation of DNA replication in eukaryotes. We have characterized two mutant alleles, mcm3-1 and mcm3-10, in Saccharomyces cerevisiae and showed that they are defective at different steps of the replication initiation process. Mcm3-10 contains a P118L substitution that compromises its interaction with Mcm5 and the recruitment of Mcm3 and Mcm7 to a replication origin. P118 is conserved between Mcm3, Mcm4, Mcm5, and Mcm7. An identical substitution of this conserved residue in Mcm5 (P83L of mcm5-bob1) strengthens the interaction between Mcm3 and Mcm5 and allows cells to enter S phase independent of Cdc7-Dbf4 kinase (Hardy, C. F., Dryga, O., Pahl, P. M. B., and Sclafani, R. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3151-3155). Mcm3-1 contains a G246E mutation that diminishes the efficiency of replication initiation (Yan, H., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160) but not its interaction with Mcm5 or recruitment of the MCM2-7 complex to replication origin. These observations indicate that Mcm3-10 is defective in a step before, and Mcm3-1 is defective in a step after the recruitment of the MCM2-7 complex to replication origins.