Mitsuhiro Shimizu

Effect of Sequence-Directed Nucleosome Disruption on Cell-Type-Specific Repression by α2/Mcm1 in the Yeast Genome

ABSTRACT In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATα cells by α2/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 α2/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 MATα 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 α-mating pheromone in MATα cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa.

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N‐terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2–105) of rice Siz1 is a four‐helix bundle with an up‐down‐extended loop‐down‐up topology, whereas the SAP domain (residues 1–111) of yeast Siz1 is comprised of five helices where the fifth helix α5 causes a significant change in the alignment of the four‐helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T‐rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses α2‐helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix α5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding. Proteins 2009.

Studies on the abnormal excretion of pyrimidine nucleosides in the urine of Yoshida ascites sarcoma-bearing rats. Increased excretion of deoxycytidine, pseudouridine and cytidine

Abstract Yoshida ascites sarcoma-bearing rats excreted significantly higher quantities of deoxycytidine and pseudouridine in urine than normal rats, with a peak 5 days after transplantation of the tumor cells, and excretion of cytidine peaking 3 or 4 days later. The contribution by the injected [14C]orotic acid to labeling of urinary deoxycytidine was 32 and 3 times higher than that by [14C]uridine or [14C]cytidine, respectively. Urinary pseudouridine was also labeled 5–6 times greater by [14C]orotic acid than by [14C]uridine or [14C]cytidine. The labeling in pseudouridine was as high as that in deoxycytidine by either [14C]orotic acid or [14C]cytidine and was about 10 times higher by [14C]uridine. Neither [14C]uracil nor [3H]thymidine resulted in any labeling of either nucleoside. [6-3H]Uridine resulted in radioactivity of urinary pseudouridine, whereas [5-3H]uridine did not. The extent of labeling by the injected [14C]orotic acid of urinary deoxycytidine and pseudouridine was almost constant, at least for several days around maximal excretion of nucleosides; this was true for each injection made 1, 3 or 5 days after tumor transplantation. This study suggests that an increase of urinary deoxycytidine and pseudouridine could be derived from not only the tumor cells but also from the host liver and that urinary pseudouridine could be synthesized by rearranging the ribose in a uridine molecule, i.e., by transferring the ribose from the nitrogen 1 position of uracil to the carbon 5 position.

5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells

5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the alpha2 repressor complex bound to its alpha2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.

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

ABSTRACT 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, whch 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 Δpho4 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.

Cryogenic coherent x-ray diffraction imaging for biological non-crystalline particles using the KOTOBUKI-1 diffraction apparatus at SACLA

We have developed an experimental apparatus named KOTOBUKI-1 for use in the coherent x-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. The apparatus allows us to collect diffraction data for frozen-hydrated specimens at 66 K and provides an experimental environment to easily transfer frozen-hydrated specimens from liquid nitrogen storage to the specimen stage for x-ray exposure. Since 2012, the apparatus has been used in the single-shot diffraction data collection of non-crystalline biological cells and cellular components with dimensions from micrometer to submicrometer using x-ray free electron lasers at SACLA. Here we report on the performance of the KOTOBUKI-1 diffraction apparatus and some structure analyses of biological cells and cellular components. Based on the present results, we also discuss the future developments of diffraction apparatus for more efficient data collection.

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma

Abstract Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [3H] tRNA extracted from E. coli B grown in the presence of [5,6-3H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [3H] tRNA. Neither [5-3H]-uridine, [5,6-3H]-UTP, nor [5,6-3H]-poly U released tritium in the present assay conditions.

5-bromodeoxyuridine induces transcription of repressed genes with disruption of nucleosome positioning.

5‐Bromodeoxyuridine (BrdU) modulates the expression of particular genes associated with cellular differentiation and senescence when incorporated into DNA instead of thymidine (dThd). To date, a molecular mechanism for this phenomenon remains a mystery in spite of a large number of studies. Recently, we have demonstrated that BrdU disrupts nucleosome positioning on model plasmids mediated by specific AT‐tracts in yeast cells. Here we constructed a cognate plasmid that can form an ordered array of nucleosomes determined by an α2 operator and contains the BAR1 gene as an expression marker gene to examine BAR1 expression in dThd‐auxotrophic MATα cells under various conditions. In medium containing dThd, BAR1 expression was completely repressed, associated with the formation of the stable array of nucleosomes. Insertion of AT‐tracts into a site of the promoter region slightly increased BAR1 expression and slightly destabilized nucleosome positioning dependent on their sequence specificity. In medium containing BrdU, BAR1 expression was further enhanced, associated with more marked disruption of nucleosome positioning on the promoter region. Disruption of nucleosome positioning seems to be sufficient for full expression of the marker gene if necessary transcription factors are supplied. Incorporation of 5‐bromouracil into the plasmid did not weaken the binding of the α2/Mcm1 repressor complex to its legitimate binding site, as revealed by an in vivo UV photofootprinting assay. These results suggest that BrdU increases transcription of repressed genes by disruption of nucleosome positioning around their promoters.

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.

Hap1p photofootprinting as an in vivo assay of repression mechanism in Saccharomyces cerevisiae

Publisher Summary This chapter explores the gene expression levels, resulting from the dynamic interplay of activators and repressors. These factors influence the basal transcriptional machinery, directly or indirectly, through interactions that govern each others activity or access to DNA. The strategy employs in vivo ultraviolet (UV) photofootprinting in order to detect activator binding with one of the most widely used yeast reporter genes, CYC1-lacZ. It has been utilized for analysis of many different repressors and repression sites. The UV photofootprinting has been widely used for analyzing the DNA structure and protein–DNA interaction in vitro and in vivo. This technique is based on the premise that, changes in DNA structure or the formation of protein–DNA complexes alter rates of dimerization of adjacent based on a particular DNA strand. The chapter also highlights that binding of Hap1p to UAS1 is detectable by in vivo UV photofootprinting. The useful feature of footprinting is to bind the Hap1p results in a positive signal, and the enhancement of formation of UV photoproducts. Thus, detection is extremely sensitive compared to a protection footprint because even limited occupancy of the UAS1 site yields a detectable signal.