Sequence-directed nucleosome-depletion is sufficient to activate transcription from a yeast core promoter in vivo.

Nucleosome-depleted regions (NDRs) (also called nucleosome-free regions or NFRs) are often found in the promoter regions of many yeast genes, and are formed by multiple mechanisms, including the binding of activators and enhancers, the actions of chromatin remodeling complexes, and the specific DNA sequences themselves. However, it remains unclear whether NDR formation per se is essential for transcriptional activation. Here, we examined the relationship between nucleosome organization and gene expression using a defined yeast reporter system, consisting of the CYC1 minimal core promoter and the lacZ gene. We introduced simple repeated sequences that should be either incorporated in nucleosomes or excluded from nucleosomes in the site upstream of the TATA boxes. The (CTG)12, (GAA)12 and (TGTAGG)6 inserts were incorporated into a positioned nucleosome in the core promoter region, and did not affect the reporter gene expression. In contrast, the insertion of (CGG)12, (TTAGGG)6, (A)34 or (CG)8 induced lacZ expression by 10-20 fold. Nucleosome mapping analyses revealed that the inserts that induced the reporter gene expression prevented nucleosome formation, and created an NDR upstream of the TATA boxes. Thus, our results demonstrated that NDR formation dictated by DNA sequences is sufficient for transcriptional activation from the core promoter in vivo.

Telomeric repeats act as nucleosome-disfavouring sequences in vivo.

Telomeric DNAs consist of tandem repeats of G-clusters such as TTAGGG and TG1-3, which are the human and yeast repeat sequences, respectively. In the yeast Saccharomyces cerevisiae, the telomeric repeats are non-nucleosomal, whereas in humans, they are organized in tightly packaged nucleosomes. However, previous in vitro studies revealed that the binding affinities of human and yeast telomeric repeat sequences to histone octamers in vitro were similar, which is apparently inconsistent with the differences in the human and yeast telomeric chromatin structures. To further investigate the relationship between telomeric sequences and chromatin structure, we examined the effect of telomeric repeats on the formation of positioned nucleosomes in vivo by indirect end-label mapping, primer extension mapping and nucleosome repeat analyses, using a defined minichromosome in yeast cells. We found that the human and yeast telomeric repeat sequences both disfavour nucleosome assembly and alter nucleosome positioning in the yeast minichromosome. We further demonstrated that the G-clusters in the telomeric repeats are required for the nucleosome-disfavouring properties. Thus, our results suggest that this inherent structural feature of the telomeric repeat sequences is involved in the functional dynamics of the telomeric chromatin structure.

Site-specific incorporation of functional components into RNA by an unnatural base pair transcription system.

Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3'-terminus. We found that the efficient incorporation into a position close to the 3'-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

Highly efficient chromatin transcription induced by superhelically curved DNA segments: the underlying mechanism revealed by a yeast system.

Superhelically curved DNA structures can strongly activate transcription in mammalian cells. However, the mechanism underlying the activation has not been clarified. We investigated this mechanism in yeast cells, using 108, 180, and 252 bp synthetic curved DNA segments. Even in the presence of nucleosomes, these DNAs activated transcription from a UAS-deleted CYC1 promoter that is silenced in the presence of nucleosomes. The fold-activations of transcription by these segments, relative to the transcription on the control that lacked such segments, were 51.4, 63.4, and 56.4, respectively. The superhelically curved DNA structures favored nucleosome formation. However, the translational positions of the nucleosomes were dynamic. The high mobility of the nucleosomes on the superhelically curved DNA structures seemed to influence the mobility of the nucleosomes formed on the promoter and eventually enhanced the access to the center region of one TATA sequence. Functioning as a dock for the histone core and allowing nucleosome sliding seem to be the mechanisms underlying the transcriptional activation by superhelically curved DNA structures in chromatin. The present study provides important clues for designing and constructing artificial chromatin modulators, as a tool for chromatin engineering.

Transcriptional repression by the Pho4 transcription factor controls the timing of SNZ1 expression.

Nutrient-sensing kinases play important roles for the yeast Saccharomyces cerevisiae to adapt to new nutrient conditions when the nutrient status changes. Our previous global gene expression analysis revealed that the Pho85 kinase, one of the yeast nutrient-sensing kinases, is involved in the changes in gene expression profiles when yeast cells undergo a diauxic shift. We also found that the stationary phase-specific genes SNZ1 and SNO1, which share a common promoter, are not properly induced when Pho85 is absent. To examine the role of the kinase in SNZ1/SNO1 regulation, we analyzed their expression during the growth of various yeast mutants, including those affecting Pho85 function or lacking the Pho4 transcription factor, an in vivo substrate of Pho85, and tested Pho4 binding by chromatin immunoprecipitation. Pho4 exhibits temporal binding to the SNZ1/SNO1 promoter to down-regulate the promoter activity, and a Deltapho4 mutation advances the timing of SNZ1/SNO1 expression. SNZ2, another member of the SNZ/SNO family, is expressed at an earlier growth stage than SNZ1, and Pho4 does not affect this timing, although Pho85 is required for SNZ2 expression. Thus, Pho4 appears to regulate the different timing of the expression of the SNZ/SNO family members. Pho4 binding to the SNZ1/SNO1 promoter is accompanied by alterations in chromatin structure, and Rpd3 histone deacetylase is required for the proper timing of SNZ1/SNO1 expression, while Asf1 histone chaperone is indispensable for their expression. These results imply that Pho4 plays positive and negative roles in transcriptional regulation, with both cases involving structural changes in its target chromatin.

In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer.

The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA-protein interactions and chromatin structure in vivo and in vitro.

A nucleosome positioned by alpha2/Mcm1 prevents Hap1 activator binding in vivo.

Nucleosome positioning has been proposed as a mechanism of transcriptional repression. Here, we examined whether nucleosome positioning affects activator binding in living yeast cells. We introduced the cognate Hap1 binding site (UAS1) at a location 24-43 bp, 29-48 bp, or 61-80 bp interior to the edge of a nucleosome positioned by alpha2/Mcm1 in yeast minichromosomes. Hap1 binding to the UAS1 was severely inhibited, not only at the pseudo-dyad but also in the peripheral region of the positioned nucleosome in alpha cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in alpha cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which alpha2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.

Effect of sequence-directed nucleosome disruption on cell-type-specific repression by alpha2/Mcm1 in the yeast genome.

In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATalpha cells by alpha2/Mcm1, acting in concert with the Ssn6-Tup1 corepressors and the Isw2 chromatin remodeling complex, and nucleosome positioning has been proposed as one mechanism of repression. However, prior studies showed that nucleosome positioning is not essential for repression by alpha2/Mcm1 in artificial reporter plasmids, and the importance of the nucleosome positioning remains questionable. We have tested the function of positioned nucleosomes through alteration of genomic chromatin at the a-cell-specific gene BAR1. We report here that a positioned nucleosome in the BAR1 promoter is disrupted in cis by the insertion of diverse DNA sequences such as poly(dA) . poly(dT) and poly(dC-dG) . poly(dC-dG), leading to inappropriate partial derepression of BAR1. Also, we show that isw2 mutation causes loss of nucleosome positioning in BAR1 in MATalpha cells as well as partial disruption of repression. Thus, nucleosome positioning is required for full repression, but loss of nucleosome positioning is not sufficient to relieve repression completely. Even though disruption of nucleosome positioning by the cis- and trans-acting modulators of chromatin has a modest effect on the level of transcription, it causes significant degradation of the alpha-mating pheromone in MATalpha cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.

Effect of histone methyltransferase gene mutations on sporulation in S. cerevisiae.

Early meiotic gene expression in Saccharomyces cerevisiae is regulated through chromatin alterations. To elucidate chromatin function in meiotic gene expression, we have studied the roles of histone methyltransferases in sporulation. Three histone lysine methyltransferases, Set1p, Set2p and Dot1p, have been identified in S. cerevisiae. We constructed a series of strains carrying set1delta set2delta and dot1delta mutations, and characterized sporulation process of these mutant strains. It was found that set1delta set2delta double and set1delta set2delta dot1delta triple mutations severely impaired spore formation. Because set1delta and set2delta affect this process additively, we suggest that Set1p and Set2p have overlapping functions in this developmental process.