Single-Molecule Analysis Reveals Linked Cycles of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast.

It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.

Discovery of an unusual copper homeostatic mechanism in yeast cells respiring on minimal medium and an unexpectedly diverse labile copper pool.

Copper is essential for all eukaryotic cells but many details of how it is trafficked within the cell and how it is homeostatically regulated remain uncertain. Here, we characterized the copper content of cytosol and mitochondria using liquid chromatography with ICP-MS detection. Chromatograms of cytosol exhibited over two dozen peaks due to copper proteins and coordination complexes. Yeast cells respiring on minimal media did not regulate copper import as media copper concentration increased; rather, they imported copper at increasing rates while simultaneously increasing the expression of metallothionein CUP1 which then sequestered most of the excessive imported copper. Peak intensities due to superoxide dismutase SOD1, other copper proteins, and numerous coordination complexes also increased, but not as drastically. The labile copper pool was unexpectedly diverse and divided into two groups. One group approximately comigrated with copper-glutathione, -cysteine, and -histidine standards; the other developed only at high media copper concentrations and at greater elution volumes. Most cytosolic copper arose from copper-bound proteins, especially CUP1. Cytosol contained an unexpectedly high percentage of apo-copper proteins and apo-coordination complexes. Copper-bound forms of non-CUP1 proteins and complexes coexisted with apo-CUP1 and with the chelator BCS. Both experiments suggest unexpectedly stable-binding copper proteins and coordination complexes in cytosol. COX17Δ cytosol chromatograms were like those of WT cells. Chromatograms of soluble mitochondrial extracts were obtained, and mitoplasting helped distinguish copper species in the intermembrane space versus in the matrix/inner membrane. Issues involving the yeast copperome, copper homeostasis, labile copper pool, and copper trafficking are discussed.

Mössbauer and LC-ICP-MS investigation of iron trafficking between vacuoles and mitochondria in vma2ΔSaccharomyces cerevisiae.

Vacuoles are acidic organelles that store FeIII polyphosphate, participate in iron homeostasis, and have been proposed to deliver iron to mitochondria for iron-sulfur cluster (ISC) and heme biosynthesis. Vma2Δ cells have dysfunctional V-ATPases, rendering their vacuoles nonacidic. These cells have mitochondria that are iron-dysregulated, suggesting disruption of a putative vacuole-to-mitochondria iron trafficking pathway. To investigate this potential pathway, we examined the iron content of a vma2Δ mutant derived from W303 cells using Mössbauer and EPR spectroscopies and liquid chromatography interfaced with inductively-coupled-plasma mass spectrometry. Relative to WT cells, vma2Δ cells contained WT concentrations of iron but nonheme FeII dominated the iron content of fermenting and respiring vma2Δ cells, indicating that the vacuolar FeIII ions present in WT cells had been reduced. However, vma2Δ cells synthesized WT levels of ISCs/hemes and had normal aconitase activity. The iron content of vma2Δ mitochondria was similar to WT, all suggesting that iron delivery to mitochondria was not disrupted. Chromatograms of cytosolic flow-through solutions exhibited iron species with apparent masses of 600 and 800 Da for WT and vma2∆, respectively. Mutant cells contained high copper concentrations and high concentrations of a species assigned to metallothionein, indicating copper dysregulation. vma2Δ cells from previously studied strain BY4741 exhibited iron-associated properties more consistent with prior studies, suggesting subtle strain differences. Vacuoles with functional V-ATPases appear unnecessary in W303 cells for iron to enter mitochondria and be used in ISC/heme biosynthesis; thus, there appears to be no direct or dedicated vacuole-to-mitochondria iron trafficking pathway. The vma2Δ phenotype may arise from alterations in trafficking of iron directly from cytosol to mitochondria.

Expression of monellin in a food-grade delivery system in Saccharomyces cerevisiae.

BACKGROUND: Genetically modified (GM) foods have caused much controversy. Construction of a food-grade delivery system is a desirable technique with presumptive impact on industrial applications from the perspective of bio-safety. The aim of this study was to construct a food-grade delivery system for Saccharomyces cerevisiae and to study the expression of monellin from the berries of the West African forest plant Dioscoreophyllum cumminsii in this system. RESULTS: A food-grade system for S. cerevisiae was constructed based on ribosomal DNA (rDNA)-mediated homologous recombination to enable high-copy-number integration of the expression cassette inserted into the rDNA locus. A copper resistance gene (CUP1) was used as the selection marker for yeast transformation. Because variants of transformants containing different copy numbers at the CUP1 locus can be readily selected after growth in the presence of elevated copper levels, we suggest that this system would prove useful in the generation of tandemly iterated gene clusters. Using this food-grade system, a single-chain monellin gene was heterologously expressed. The yield of monellin reached a maximum of 675 mg L(-1) . CONCLUSION: This system harbors exclusively S. cerevisiae DNA with no antibiotic resistance genes, and it should therefore be appropriate for safe use in the food industry. Monellin was shown to be expressed in this food-grade delivery system. To our knowledge, this is the first report so far on expression of monellin in a food-grade expression system in S. cerevisiae.

Enhancing high-efficient cadmium biosorption of Escherichia coli via cell surface displaying metallothionien CUP1.

Cadmium (Cd) is one of the common heavy metal pollutants in soil, which can induce various diseases and pose a serious threat to human health. Metallothioneins (MTs) are well-known for their excellent metal binding ability due to a high content of cysteine, which has great potential for heavy metal chelation. In this study, we used the Escherichia coli (E. coli) surface display system LPP-OmpA to construct a recombinant plasmid pBSD-LCF encoding LPP-OmpA-CUP1-Flag fusion protein. Then we displayed the metallothionein CUP1 from Saccharomyces cerevisiae on E. coli DH5α surface for Cd removing. The feasibility of surface display of metallothionein CUP1 in recombinant E. coli DH5α (pBSD-LCF) by Lpp-OmpA system was proved by flow cytometry and western blot analysis, and the specificity of the fusion protein in the recombinant strain was also verified. The results showed that the Cd2+ resistance capacity of DH5α (pBSD-LCF) was highly enhanced by about 200%. Fourier-transform infrared spectroscopy showed that sulfhydryl and sulfonyl groups were involved in Cd2+ binding to cell surface of DH5α (pBSD-LCF). Meanwhile, Cd removal rate by DH5α (pBSD-LCF) was promoted to 95.2%. Thus, the recombinant strain E. coli DH5α (pBSD-LCF) can effectively chelate environmental metals, and the cell surface expression of metallothionein on E. coli can provide new ideas and directions for heavy metals remediation.

The N-Terminal Tail of Histone H3 Regulates Copper Homeostasis in Saccharomyces cerevisiae.

Copper homeostasis is crucial for various cellular processes. The balance between nutritional and toxic copper levels is maintained through the regulation of its uptake, distribution, and detoxification via antagonistic actions of two transcription factors, Ace1 and Mac1. Ace1 responds to toxic copper levels by transcriptionally regulating detoxification genes CUP1 and CRS5 Cup1 metallothionein confers protection against toxic copper levels. CUP1 gene regulation is a multifactorial event requiring Ace1, TATA-binding protein (TBP), chromatin remodeler, acetyltransferase (Spt10), and histones. However, the role of histone H3 residues has not been fully elucidated. To investigate the role of the H3 tail in CUP1 transcriptional regulation, we screened the library of histone mutants in copper stress. We identified mutations in H3 (K23Q, K27R, K36Q, Δ5-16, Δ13-16, Δ13-28, Δ25-28, Δ28-31, and Δ29-32) that reduce CUP1 expression. We detected reduced Ace1 occupancy across the CUP1 promoter in K23Q, K36Q, Δ5-16, Δ13-28, Δ25-28, and Δ28-31 mutations correlating with the reduced CUP1 transcription. The majority of these mutations affect TBP occupancy at the CUP1 promoter, augmenting the CUP1 transcription defect. Additionally, some mutants displayed cytosolic protein aggregation upon copper stress. Altogether, our data establish previously unidentified residues of the H3 N-terminal tail and their modifications in CUP1 regulation.

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats.

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

Massive Amplification at an Unselected Locus Accompanies Complex Chromosomal Rearrangements in Yeast.

Gene amplification has been observed in different organisms in response to environmental constraints, such as limited nutrients or exposure to a variety of toxic compounds, conferring them with specific phenotypic adaptations via increased expression levels. However, the presence of multiple gene copies in natural genomes has generally not been found in the absence of specific functional selection. Here, we show that the massive amplification of a chromosomal locus (up to 880 copies per cell) occurs in the absence of any direct selection, and is associated with low-order amplifications of flanking segments in complex chromosomal alterations. These results were obtained from mutants with restored phenotypes that spontaneously appeared from genetically engineered strains of the yeast Saccharomyces cerevisiae suffering from severe fitness reduction. Grossly extended chromosomes (macrotene) were formed, with complex structural alterations but sufficient stability to propagate unchanged over successive generations. Their detailed molecular analysis, including complete genome sequencing, identification of sequence breakpoints, and comparisons between mutants, revealed novel mechanisms causing their formation, whose combined action underlies the astonishing dynamics of eukaryotic chromosomes and their consequences.

Too much of a good thing: the unique and repeated paths toward copper adaptation.

Copper is a micronutrient essential for growth due to its role as a cofactor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multilayered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1-1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1-1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient.