The mitotic Clb cyclins are required to alleviate HIR-mediated repression of the yeast histone genes at the G1/S transition.

The histone genes are an important group of cell cycle regulated genes whose transcription is activated during the G1/S transition and repressed in early G1, late S, and G2/M. The HIR complex, comprised of Hir1, Hir2, Hir3 and Hpc2, regulates three of the four histone gene loci. While relief of repression at the G1/S boundary involves the HIR complex, as well as other cofactors, the mechanism by which this derepression occurs remains unknown. To better understand how transcriptional repression contributes to periodic expression in the cell cycle, we sought to identify the cell cycle signals required to alleviate HIR-mediated repression of the histone genes. By measuring histone gene transcription in strains with various combinations of clb mutations, we found that the mitotic Clb1/Clb2 cyclins are required to alleviate Hir-mediated repression during the G1/S transition and that Clb2 physically interacts with the HIR complex. While the HIR complex regulates histone gene transcription in combination with two other histone H3/H4 chaperones, Asf1 and Rtt106, our data demonstrate that the mitotic Clb cyclins are necessary to specifically alleviate the repressive action of the HIR complex itself in order to allow proper expression of the histone genes in late G1/early S phase.

Separation-of-function mutation in HPC2, a member of the HIR complex in S. cerevisiae, results in derepression of the histone genes but does not confer cryptic TATA phenotypes.

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63-79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452-480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.

Development of a gene silencing DNA vector derived from a broad host range geminivirus.

BACKGROUND: Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. RESULTS: The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. CONCLUSION: The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

Transcriptional regulation of yeast phospholipid biosynthetic genes.

The last several years have been witness to significant developments in understanding transcriptional regulation of the yeast phospholipid structural genes. The response of most phospholipid structural genes to inositol is now understood on a mechanistic level. The roles of specific activators and repressors are also well established. The knowledge of specific regulatory factors that bind the promoters of phospholipid structural genes serves as a foundation for understanding the role of chromatin modification complexes. Collectively, these findings present a complex picture for transcriptional regulation of the phospholipid biosynthetic genes. The INO1 gene is an ideal example of the complexity of transcriptional control and continues to serve as a model for studying transcription in general. Furthermore, transcription of the regulatory genes is also subject to complex and essential regulation. In addition, databases resulting from a plethora of genome-wide studies have identified regulatory signals that control one of the essential phospholipid biosynthetic genes, PIS1. These databases also provide significant clues for other regulatory signals that may affect phospholipid biosynthesis. Here, we have tried to present a complete summary of the transcription factors and mechanisms that regulate the phospholipid biosynthetic genes.

Genomic analysis of the Opi- phenotype.

Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi(-) phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi(-) mutants. In total, 89 Opi(-) mutants were identified, of which 7 were previously known to have the Opi(-) phenotype. The Opi(-) mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p-Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi(-) phenotype because of a buildup of PA. However, in this case the Opi(-) phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi(-) collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi(-) mutants were found to affect the UPR (yhi9, ede1, and vps74).