Upstream Activation Sequence Research Articles

Drosophila functional screening of de novo variants in autism uncovers damaging variants and facilitates discovery of rare neurodevelopmental diseases.

SUMMARYIndividuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through “humanization” rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Programmable Synthetic Upstream Activating Sequence Library for Fine-Tuning Gene Expression Levels in Saccharomyces cerevisiae.

A wide dynamic range of promoters is necessary for fine-tuning transcription levels. However, weak intensity and narrow dynamic range limit transcriptional regulation via constitutive promoters. The upstream activation sequence (UAS) located upstream of the core promoter is a crucial region that could obviously enhance promoter strength. Herein, we created a random mutagenesis library consisting of 330 different variants based on the UAS of the TDH3 promoter with an ∼37-fold dynamic range by error-prone polymerase chain reaction (PCR) and obtained strong intensity mutant UAS, which was ∼12-fold greater than the wild-type UASTDH3. Analysis of the mutant library revealed 15 strength-enhancing sites and their corresponding bases of the UASTDH3 regions, which provided the impetus for a synthetic library. The resulting 32 768 mutant UAS library was constructed by permutation and combination of the bases of the 15 enhancing sites. To characterize the library, a strength prediction model was built by correlating DNA structural features and UAS strength, which provided a model between UAS sequence and intensity. Following characterization, the UAS library was applied to precisely regulate gene expression in the production of β-carotene, proving that the UAS library would be a useful tool for gene tuning in metabolic engineering. In summary, we designed, constructed, and characterized a UAS library that facilitated precise tuning of transcription levels of target proteins.

Upstream Activation Sequence Can Function as an Insulator for Chromosomal Regulation of Heterologous Pathways Against Position Effects in Saccharomyces cerevisiae.

Metabolic engineering of microbial cell factories through integrating the heterologous synthetic pathway into the chromosome is most commonly used for industrial applications. However, the position of the foreign gene in the chromosome can affect its transcriptional level. As a microorganism that is generally regarded as safe (GRAS) and commonly applied in industrial manufacture with large-scale operations, Saccharomyces cerevisiae is also confronted with this position effect. In this study, we characterized 12 different chromosome sites by inserting the lycopene biosynthetic pathway as a reporter cassette. Due to the different integration loci, the gene transcription and lycopene yield exhibited more than 58-fold and 3.8-fold differences, respectively. Furthermore, changing the gene order also revealed a remarkable influence (30-fold and 14-fold) on gene transcription and lycopene yield. Besides, the upstream activation sequence of a strong promoter (defined as an insulator) in S. cerevisiae could reduce the impact by gene order, and increased the gene transcription (tenfold) and lycopene yield (sevenfold). Taken together, our results demonstrated that gene order and insulator affected gene transcription and heterogeneous biosynthesis, opening the opportunity to regulate gene transcription by insulator against position effect in S. cerevisiae.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801bp,and -284 and -108bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (PPDH, PPYK, PFBA, PPGM, PGLK, PTRI, PGPI, PADH1, PTEF1 and PGCW14) were obtained with the activity range of 13%–130% of the common promoter PGAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which PPDH and PGCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced PICL1, rhamnose-induced PLRA3 and PLRA4, and a bidirectional promoter (PMal-PPer) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter PLRA3 by 4.7–10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

Measuring the buffering capacity of gene silencing in Saccharomyces cerevisiae

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known.ResultsHere, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources.ConclusionsIn this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

Cytokine exposure mediates transcriptional activation of the orphan nuclear receptor Nur77 in hematopoietic cells

The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells.

Reciprocal stabilization of transcription factor binding integrates two signaling pathways to regulate fission yeast fbp1 transcription.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

BackgroundIn biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.ResultsIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.ConclusionWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Synthetic Promoters from Strawberry Vein Banding Virus (SVBV) and Dahlia Mosaic Virus (DaMV).

We have constructed two intra-molecularly shuffled promoters, namely S100 and D100. The S100 recombinant promoter (621bp) was generated by ligation of 250bp long upstream activation sequence (UAS) of Strawberry vein banding virus (SV10UAS; - 352 to - 102 relative to TSS) with its 371bp long TATA containing core promoter domain (SV10CP; - 352 to + 19). Likewise, 726bp long D100 promoter was constructed by fusion of 170bp long UAS of Dahlia mosaic virus (DaMV14UAS; - 203 to - 33) with its 556bp long core promoter domain (DaMV4CP; - 474 to + 82). S100 and D100 promoters showed 1.8 and 2.2 times stronger activities than that of the CaMV35S promoter. The activity of the promoters is comparable to that of the CaMV35S2 promoter. Transcript analysis employing qRT-PCR and histochemical assays supported the above findings. Abscisic acid and salicylic acid induce the activity of the D100 promoter. Leaf protein obtained from Nicotiana tabacum plant expressing NSD2 gene (Nigella sativa L. defensin 2) driven by the D100 promoter showed antifungal activity against Alternaria alternata and Phoma exigua var. exigua and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Strong S100 and D100 promoters have potential to become efficient candidates for plant metabolic engineering and molecular pharming.

Synthetic promoters from blueberry red ringspot virus (BRRV).

We analyzed the synthetic full-length transcript promoter of Blueberry red ringspot virus (BRRV) and developed two chimeric promoters (MBR3 and FBR3). Transcriptional activities of these chimeric promoters were found equivalent to that of the CaMV35S2 promoter. Chimeric promoters driven plant-derived PaDef protein showed high antimicrobial activities against several pathogens. Blueberry red ringspot virus (BRRV) is a pararetrovirus under the genus, Soymovirus belongs to the Caulimoviridae family. We have made a synthetic version of the BRRV-Flt promoter and analyzed its activity in detail. A 372bp promoter fragment BR3 (- 212 to + 160) showed the strongest transcriptional activity compared with other fragments in both transient and transgenic assays; its activity was found near equivalent to that of the CaMV35S promoter. We constructed two chimeric promoters; MBR3 and FBR3 by fusing the UASs (Upstream activation sequences) of Mirabilis mosaic virus (MUAS; - 297 to - 38; 335bp) and Figwort mosaic virus (FUAS; - 249 to - 54; 303bp) respectively to the core promoter domain of BR3 (BR3; - 212 to + 160; 372bp). The activities of MBR3 and FBR3 promoters were found equivalent to that of the activity of the CaMV35S2 promoter and approximately 4.0 (four) times stronger than that of the CaMV35S promoter. Histochemical and fluorometric GUS assays confirmed the above observation. The transcriptional efficacies of these recombinant promoters were tested by evaluating the antibacterial and antifungal activities of recombinant plant-derived antimicrobial peptide Persea americana var. drymifolia defensin (PaDef) driven under these promoters. Bioassays showed promising antifungal activities of the plant made PaDef against Alternaria alternata and antibacterial property against Gram-positive (S. aureus and R. fascians) and Gram-negative bacteria (E. coli and P. aeruginosa). Based upon the above results, MBR3 and FBR3 could be useful promoters for plant genetic engineering and can become useful substitutes for the widely used CaMV35S2 promoter in plant biology.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Ameliorative influence of atorvastatin in transgenic Drosophila Melanogaster model of neurodegenerative diseases

Background: The common features in the pathogenesis of Alzheimer's disease (AD) and Parkinson disease (PD) (two most common neurodegenerative diseases) are chronic and progressive aggregation and accumulation of misfolded proteins (amyloid-beta and tau proteins in AD as well as α-synuclein in PD) leading to the destruction of vulnerable neurons and synaptic connections and ultimately neuronal cell death brain mass loss. Despite our knowledge of the molecular mechanisms implicated in AD and PD pathogenesis and primary target of therapeutic intervention being the misfolded protein aggregates, no effiffifficient treatments are available. The fruit flfly, Drosophila melanogaster (Drosophila), is a valuable model organism for neurodegenerative disease owing to its short lifespan and plethora of genetic tools for exquisite targeted manipulation of the genome. Thus, in this study the protective action of atorvastatin on genetic model of AD and PD in mice. Methods: To model PD and AD in Drosophila, the bipartite system of GAL4 transcriptional activator was placed under a cell-type specifific promoter; embryonic lethal abnormal visual system GAL4 (ELAV-GAL4) or dopa decarboxylase (Ddc-GAL4) for expression of amyloid-beta (Aβ42) or α-synuclein, respectively, under the control of the upstream activating sequence (UAS) in Drosophila. The flflies were was either maintained on media supplemented with vehicle or atorvastatin (85, 170 or 340µM; HMG-CoA reductase inhibitor – antihyperlipidemic drug). The effffect of treatments on larva motility, climbing activity, fecundity and lifespan were recorded. Results: Supplementation of flfly media with difffferent concentration of atorvastatin ameliorated the deficits in larva motility and climbing activity. Moreover, supplementation of flfly media with atorvastatin prolonged the survival of drosophila but atorvastatin (384µM) reduced fecundity. Conclusion: Findings from this study showed that atorvastatin improved spontaneous motor activity and prolonged lifespan in Drosophila possibly through reduction of misfolded protein aggregates.

Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila.

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency. RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II

The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM–cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.

Construction and characterization of a zinc-inducible gene expression vector in fission yeast.

Gene expression vectors are useful and important tools that are commonly used in a variety of experiments, including expression of foreign genes, functional analysis of genes of interest and complementation experiments. In this study, a hybrid promoter, combining the adh1+ upstream activating sequence (UAS) of fission yeast and the GAL10 core promoter of budding yeast, was constructed to enable high level expression depending on the presence of zinc in culture medium for fission yeast. When the hybrid promoter was cloned on the multicopy plasmid, it was fully induced and repressed within 10 h in the presence and absence of zinc, respectively. The kinetics of induction and reduction were similar to those of the endogenous adh1+ mRNA. In contrast, native adh1+ promoter lost its tight repression in zinc-depleted condition when it was cloned on the plasmid. Because adh1+ UAS-specific transcription factors have not yet been identified, we identified UAS elements involved in zinc sensing by characterizing this hybrid promoter. We also found that the expression level increased by the TATA box mutation, GATAA, in the presence of zinc.

Facilitating fructose-driven metabolism exerts a protective effect on anoxic stress in Drosophila.

Hypoxic stress is linked to various cardiovascular disorders (e.g., stroke, myocardial infarction), mediated, at least in part, by a reduction in ATP synthesis. Fructose-driven glycolysis is proposed as an alternative pathway capable of sustaining ATP production even under anoxic conditions. Here, we tested the hypothesis that facilitating fructose-driven metabolism exerts a protective effect against anoxic stress in Drosophila. Genetically modified flies with the human fructose transporter (GluT5) and ketohexokinase (KHK) genes downstream of upstream activating sequence (UAS) were constructed. The GAL4-UAS system was confirmed to: (i) increase the expression of GluT5 and KHK in a tissue-specific and a time-dependent manner (i.e., whole flies [with Act5c-gene switch GAL4 driver], neurons [with elav-gene switch GAL4 driver]) and (ii) reduce mortality of flies when placed under anoxic stress. Taken together, these data suggest that increasing fructose metabolism may be a clinically relevant approach to minimize hypoxia-induced cellular damage.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae.

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiaeHO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.