Showing papers on "Upstream activating sequence published in 2017"

New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica

Abstract: The oleaginous yeast Yarrowia lipolytica is increasingly used as alternative cell factory for the production of recombinant proteins. At present, several promoters with different strengths have been developed based either on the constitutive pTEF promoter or on oleic acid inducible promoters such as pPOX2 and pLIP2. Although these promoters are highly efficient, there is still a lack of versatile inducible promoters for gene expression in Y. lipolytica. We have isolated and characterized the promoter of the EYK1 gene coding for an erythrulose kinase. pEYK1 induction was found to be impaired in media supplemented with glucose and glycerol, while the presence of erythritol and erythrulose strongly increased the promoter induction level. Promoter characterization and mutagenesis allowed the identification of the upstream activating sequence UAS1EYK1. New hybrid promoters containing tandem repeats of either UAS1XPR2 or UAS1EYK1 were developed showing higher expression levels than the native pEYK1 promoter. Furthermore, promoter strength was improved in a strain carrying a deletion in the EYK1 gene, allowing thus the utilization of erythritol and erythrulose as free inducer. Novel tunable and regulated promoters with applications in the field of heterologous protein production, metabolic engineering, and synthetic biology have been developed, thus filling the gap of the absence of versatile inducible promoter in the yeast Y. lipolytica.

Heat Shock Protein Genes Undergo Dynamic Alteration in Their Three-Dimensional Structure and Genome Organization in Response to Thermal Stress.

Abstract: Three-dimensional (3D) chromatin organization is important for proper gene regulation, yet how the genome is remodeled in response to stress is largely unknown. Here, we use a highly sensitive version of chromosome conformation capture in combination with fluorescence microscopy to investigate Heat Shock Protein (HSP) gene conformation and 3D nuclear organization in budding yeast. In response to acute thermal stress, HSP genes undergo intense intragenic folding interactions that go well beyond 5'-3' gene looping previously described for RNA polymerase II genes. These interactions include looping between upstream activation sequence (UAS) and promoter elements, promoter and terminator regions, and regulatory and coding regions (gene "crumpling"). They are also dynamic, being prominent within 60 s, peaking within 2.5 min, and attenuating within 30 min, and correlate with HSP gene transcriptional activity. With similarly striking kinetics, activated HSP genes, both chromosomally linked and unlinked, coalesce into discrete intranuclear foci. Constitutively transcribed genes also loop and crumple yet fail to coalesce. Notably, a missense mutation in transcription factor TFIIB suppresses gene looping, yet neither crumpling nor HSP gene coalescence is affected. An inactivating promoter mutation, in contrast, obviates all three. Our results provide evidence for widespread, transcription-associated gene crumpling and demonstrate the de novo assembly and disassembly of HSP gene foci.

A Strong Hybrid Fatty Acid Inducible Transcriptional Sensor Built From Yarrowia lipolytica Upstream Activating and Regulatory Sequences

Abstract: The engineering of Yarrowia lipolytica to accumulate lipids with high titers and productivities has been enabled with a handful of constitutive promoters for pathway engineering. However, the development of promoters that are both strong and lipid responsive could greatly benefit the bioproduction efficiency of lipid-derived oleochemicals in oleaginous yeast. In this study, a fatty acid regulated hybrid promoter for use in Y. lipolytica is engineered. A 200 bp upstream regulatory sequence in the peroxisomal acyl CoA oxidase 2 (POX2) promoter is identified. Further analysis of the promoter sequence reveal a regulatory sequence, that when used in tandem repeats, lead to a 48-fold induction of gene expression relative to glucose and fourfold higher than the native POX2 promoter. To date, this is the strongest inducible promoter reported in Y. lipolytica. Taken together, the results show that it is possible to engineer strong promoters that retain strong inducibility. These types of promoters will be useful in controlling metabolism and as fatty acid sensors.

A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes.

Abstract: The tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hybrid promoter pGUKd displayed dramatically increased fluorescence intensity at pH 2.5, thus verifying that pGUKd is a low-pH inducible promoter. The promoter pGUKd was then used to express the D-xylose dehydrogenase gene xylB, resulting in increased expression levels of xylB at low pH. The recombinant protein exhibited higher specific activities under lower pH conditions and produced 38 g/l D-xylonate at pH 2.5. This rate is much higher than that produced by fermentation at pH 5.5. These results suggest that the novel hybrid promoter pGUKd functions to direct the production of D-xylonate at low pH, and we provide a candidate genetic tool for the stress tolerant yeast C. glycerinogenes.

Synthetic regulatory RNAs selectively suppress the progression of bladder cancer.

Abstract: The traditional treatment for cancer is lack of specificity and efficacy. Modular synthetic regulatory RNAs, such as inhibitive RNA (iRNA) and active RNA (aRNA), may overcome these limitations. Here, we synthesize a new iRNA to bind the upstream activating sequence (UAS) of a minimal promoter that drives expression of artificial miRNAs (amiRNAs) targeting MYC, which represses the binding interaction between UAS and GAL4 fusion protein (GAL4-VP64) in GAL4/UAS system. The aRNA driven by a tumor-specific mutant human telomerase reverse transcriptase (hTERT) promoter is created to interact with iRNA to expose UAS again in bladder cancer. Without the aRNA, mRNA and protein levels of MYC, cell growth, cell apoptosis and cell migration were no significance in two bladder cancer cell lines, T24 and 5637, and human foreskin fibroblast (HFF) cells. The aRNA significantly inhibited the expression of MYC in mRNA and protein levels, as well as the proliferation and migration of the cancer cells, but not in HFF cells. These results indicated that regulatory RNAs selectively controlled the expression of amiRNAs and ultimately suppress the progression of bladder cancer cells without affecting normal cells. Synthetic regulatory RNAs might be a selective therapeutic approach for bladder cancer.

SAGA mediates transcription from the TATA-like element independently of Taf1p/TFIID but dependent on core promoter structures in Saccharomyces cerevisiae.

Abstract: In Saccharomyces cerevisiae, core promoters of class II genes contain a TATA element, either a TATA box (TATA[A/T]A[A/T][A/G]) or TATA-like element (1 or 2 bp mismatched version of the TATA box). The TATA element directs the assembly of the preinitiation complex (PIC) to ensure accurate transcriptional initiation. It has been proposed the PIC is assembled by two distinct pathways in which TBP is delivered by TFIID or SAGA, leading to the widely accepted model that these complexes mediate transcription mainly from TATA-like element- or TATA box-containing promoters, respectively. Although both complexes are involved in transcription of nearly all class II genes, it remains unclear how efficiently SAGA mediates transcription from TATA-like element-containing promoters independently of TFIID. We found that transcription from the TATA box-containing AGP1 promoter was greatly stimulated in a Spt3p-dependent manner after inactivation of Taf1p/TFIID. Thus, this promoter provides a novel experimental system in which to evaluate SAGA-mediated transcription from TATA-like element(s). We quantitatively measured transcription from various TATA-like elements in the Taf1p-dependent CYC1 promoter and Taf1p-independent AGP1 promoter. The results revealed that SAGA could mediate transcription from at least some TATA-like elements independently of Taf1p/TFIID, and that Taf1p-dependence or -independence is highly robust with respect to variation of the TATA sequence. Furthermore, chimeric promoter mapping revealed that Taf1p-dependence or independence was conferred by the upstream activating sequence (UAS), whereas Spt3p-dependent transcriptional stimulation after inactivation of Taf1p/TFIID was specific to the AGP1 promoter and dependent on core promoter regions other than the TATA box. These results suggest that TFIID and/or SAGA are regulated in two steps: the UAS first specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides the pertinent factor to conduct transcription in an appropriate manner.

An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells.

Abstract: The current therapies for treating tumors are lacking in efficacy and specificity. Synthetic biology principles may bring some new possible methods for curing cancer. Here we present a synthetic logic circuit based on the CRISPR/Cas9 system. The CRISPR/Cas9 technology has been applied in many biological fields, including cancer research. In this study, the expression of Cas9 nuclease was controlled indirectly by an enhanced hTERT promoter using the GAL4/upstream activating sequence (UAS) binding system. Cas9 was driven by 5XUAS, single guide RNA (sgRNA) was used to target mutant or wild-type HRAS, and the fusion gene GAL4-P65 was driven by the enhanced hTERT promoter. The system was tested in bladder cancer cells (T24 and 5637) and the results showed that the enhanced hTERT promoter could drive the expression of GAL4-P65 in these bladder cancer cell lines. Then all these devices were packed into lentivirus and the results of quantitative real-time PCR showed that the mRNA expression level of HRAS was selectively inhibited in the T24 and 5637 cells. The results of functional experiments suggested that the proliferation, cell migration and invasion were selectively suppressed, and that the apoptosis rate was increased in bladder cancer cells but not in human foreskin fibroblasts (HFF). In conclusion, we successfully constructed an enhanced hTERT promoter-driven CRISPR/Cas9 system and data showed that it could selectively suppress the progression of bladder cancer cells.

Zap1-dependent transcription from an alternative upstream promoter controls translation of RTC4 mRNA in zinc-deficient Saccharomyces cerevisiae.

Abstract: Maintaining zinc homeostasis is an important property of all organisms. In the yeast Saccharomyces cerevisiae, the Zap1 transcriptional activator is a central player in this process. In response to zinc deficiency, Zap1 activates transcription of many genes and consequently increases accumulation of their encoded proteins. In this report, we describe a new mechanism of Zap1-mediated regulation whereby increased transcription of certain target genes results in reduced protein expression. Transcription of the Zap1-responsive genes RTC4 and RAD27 increases markedly in zinc-deficient cells but, surprisingly, their protein levels decrease. We examined the underlying mechanism further for RTC4 and found that this unusual regulation results from altered transcription start site selection. In zinc-replete cells, RTC4 transcription begins near the protein-coding region and the resulting short transcript leader allows for efficient translation. In zinc-deficient cells, RTC4 RNA with longer transcript leaders are expressed that are not efficiently translated due to the presence of multiple small open reading frames upstream of the coding region. This regulation requires a potential Zap1 binding site located farther upstream of the promoter. Thus, we present evidence for a new mechanism of Zap1-mediated gene regulation and another way that this activator protein can repress protein expression.

Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells.

Abstract: Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell’s lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naive and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262 bp upstream of the TATA box. Additional positive regulatory elements are located between −1200 and −2406 bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between −2255 and −2269 bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760 bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.

Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiae.

Abstract: During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.

Specific protein binding to far upstream activating sequences in polymerase II promoters (Saccharomyces cerevisiae/GAIA gene/GALl, GAL7, and GALIO promoters/nitrocellulose filter binding/DNA-protection mapping)

Abstract: A binding activity specific for the upstream activating sequence of the GALJ-GAL1O promoter of Saccha- romyces cerevisiae has been purified 220-fold on the basis of a nitrocellulose filter-binding assay. The binding activity is en- riched in a nuclear preparation and is likely to be the GAL4 gene product. DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence. A nearly identical mapping pattern is obtained with the coordinately regulated GAL7 promoter. The four 30-base- pair regions of binding in the two promoters are closely homologous, with a core consensus sequence of C-GS T C-A- A-C-A-G-T-G-C-T-C-C-G-A-A-C-G-A-T. A synthetic oligo- nucleotide with such a sequence competes with the upstream activating sequence in the binding reaction.

A zinc-finger fusion protein refines Gal4-defined neural circuits

Abstract: The analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species—most notably Drosophila, neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4–UASG. One limitation of Gal4–UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system’s key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 (‘Zal1’) was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1–UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision.