Modifying Transcription Factors Research Articles

Metabolic engineering of artificially modified transcription factor SmMYB36-VP16 for high-level production of tanshinones and phenolic acids

Tanshinones and phenolic acids are the two main chemical constituents in Salvia miltiorrhiza, which are used clinically for the treatment of hypertension, coronary heart disease, atherosclerosis, and many other diseases, and have broad medicinal value. The efficient synthesis of the target products of these two metabolites in isolated plant tissues cannot be achieved without the regulation and optimization of metabolic pathways, and transcription factors play an important role as common regulatory elements in plant tissue metabolic engineering. However, most of the regulatory effects are specific to one class of metabolites, or an opposing regulation of two classes of metabolites exists. In this study, an artificially modified transcription factor, SmMYB36-VP16, was constructed to enhance tanshinones and phenolic acids in Salvia miltiorrhiza hair roots simultaneously. Further in combination with the elicitors dual-screening technique, by applying the optimal elicitors screened, the tanshinones content in the transgenic hairy roots of Salvia miltiorrhiza reached 6.44 mg/g DW, which was theoretically 6.08-fold that of the controls without any treatment, and the content of phenolic acids reached 141.03 mg/g DW, which was theoretically 5.05-fold that of the controls without any treatment. The combination of artificially modified transcriptional regulatory and elicitors dual-screening techniques has facilitated the ability of plant isolated tissue cell factories to produce targeted medicinal metabolites. This strategy could be applied to other species, laying the foundation for the production of potential natural products for the medicinal industry.

A statistical approach for identifying single nucleotide variants that affect transcription factor binding

Non-coding variants located within regulatory elements may alter gene expression by modifying transcription factor (TF) binding sites, thereby leading to functional consequences. Different TF models are being used to assess the effect of DNA sequence variants, such as single nucleotide variants (SNVs). Often existing methods are slow and do not assess statistical significance of results. We investigated the distribution of absolute maximal differential TF binding scores for general computational models that affect TF binding. We find that a modified Laplace distribution can adequately approximate the empirical distributions. A benchmark on invitro and invivo datasets showed that our approach improves upon an existing method in terms of performance and speed. Applications on eQTLs and on a genome-wide association study illustrate the usefulness of our statistics by highlighting cell type-specific regulators and target genes. An implementation of our approach is freely available on GitHub and as bioconda package.

Chromatin accessibility and H3K9me3 landscapes reveal long-term epigenetic effects of fetal-neonatal iron deficiency in rat hippocampus

BackgroundIron deficiency (ID) during the fetal-neonatal period results in long-term neurodevelopmental impairments associated with pervasive hippocampal gene dysregulation. Prenatal choline supplementation partially normalizes these effects, suggesting an interaction between iron and choline in hippocampal transcriptome regulation. To understand the regulatory mechanisms, we investigated epigenetic marks of genes with altered chromatin accessibility (ATAC-seq) or poised to be repressed (H3K9me3 ChIP-seq) in iron-repleted adult rats having experienced fetal-neonatal ID exposure with or without prenatal choline supplementation.ResultsFetal-neonatal ID was induced by limiting maternal iron intake from gestational day (G) 2 through postnatal day (P) 7. Half of the pregnant dams were given supplemental choline (5.0 g/kg) from G11–18. This resulted in 4 groups at P65 (Iron-sufficient [IS], Formerly Iron-deficient [FID], IS with choline [ISch], and FID with choline [FIDch]). Hippocampi were collected from P65 iron-repleted male offspring and analyzed for chromatin accessibility and H3K9me3 enrichment. 22% and 24% of differentially transcribed genes in FID- and FIDch-groups, respectively, exhibited significant differences in chromatin accessibility, whereas 1.7% and 13% exhibited significant differences in H3K9me3 enrichment. These changes mapped onto gene networks regulating synaptic plasticity, neuroinflammation, and reward circuits. Motif analysis of differentially modified genomic sites revealed significantly stronger choline effects than early-life ID and identified multiple epigenetically modified transcription factor binding sites.ConclusionsThis study reveals genome-wide, stable epigenetic changes and epigenetically modifiable gene networks associated with specific chromatin marks in the hippocampus, and lays a foundation to further elucidate iron-dependent epigenetic mechanisms that underlie the long-term effects of fetal-neonatal ID, choline, and their interactions.

Modulating the m6A Modified Transcription Factor GATA6 Impacts Epithelial Cytokines in Acute Lung Injury.

The methylation of the N6-position of adenosine (m6A) has been found to be associated with the inflammatory response. We hypothesize that m6A modification plays a role in the inflammation of airway epithelial cells during lung inflammation. However, the precise changes and functions of m6A modification in airway epithelial cells in acute lung injury (ALI) are not well understood. Here we report that RNA Methyltransferase 3 (METTL3)-mediated m6A of GATA6 mRNA inhibits ALI and the secretion of pro-inflammatory cytokine in airway epithelial cells. The expression of METTL3 and m6A levels decrease in lung tissues of ALI-mice. In co-cultures, peripheral blood monocytes secreted tumor necrosis factor α (TNFα) which reduces METTL3 and m6A levels in human bronchial epithelial cell line (BEAS-2B). Knockdown of METTL3 promotes IL-6 and TNFα release of BEAS-2B cells. Conversely, overexpression of METTL3 increases total RNA m6A level and reduces the levels of pro-inflammatory cytokines TNFα, transforming growth factor-β (TGFβ), and thymic stromal lymphopoietin (TSLP). Elevating METTL3 in mouse lungs prevented LPS-induced ALI and reduced the synthesis of pro-inflammatory cytokines. Mechanistically, sequencing and functional analysis show that METTL3 catalyzes m6A in the 3'UTR of GATA6 read by YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2) and triggers mRNA degradation. GATA6 knockdown rescues TNFα induced inflammatory cytokine secretion of epithelial cells, indicating that GATA6 is a main substrate of METTL3 in airway epithelial cells. Overall, this study provides evidence of a novel role for METTL3 in the inflammatory cytokine release of epithelial cells and provide an innovative therapeutic target for ALI.

CVD-associated SNPs with regulatory potential reveal novel non-coding disease genes

BackgroundCardiovascular diseases (CVDs) are the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) appearing in non-coding genomic regions in CVDs. The SNPs may alter gene expression by modifying transcription factor (TF) binding sites and lead to functional consequences in cardiovascular traits or diseases. To understand the underlying molecular mechanisms, it is crucial to identify which variations are involved and how they affect TF binding.MethodsThe SNEEP (SNP exploration and analysis using epigenomics data) pipeline was used to identify regulatory SNPs, which alter the binding behavior of TFs and link GWAS SNPs to their potential target genes for six CVDs. The human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs), monoculture cardiac organoids (MCOs) and self-organized cardiac organoids (SCOs) were used in the study. Gene expression, cardiomyocyte size and cardiac contractility were assessed.ResultsBy using our integrative computational pipeline, we identified 1905 regulatory SNPs in CVD GWAS data. These were associated with hundreds of genes, half of them non-coding RNAs (ncRNAs), suggesting novel CVD genes. We experimentally tested 40 CVD-associated non-coding RNAs, among them RP11-98F14.11, RPL23AP92, IGBP1P1, and CTD-2383I20.1, which were upregulated in hiPSC-CMs, MCOs and SCOs under hypoxic conditions. Further experiments showed that IGBP1P1 depletion rescued expression of hypertrophic marker genes, reduced hypoxia-induced cardiomyocyte size and improved hypoxia-reduced cardiac contractility in hiPSC-CMs and MCOs.ConclusionsIGBP1P1 is a novel ncRNA with key regulatory functions in modulating cardiomyocyte size and cardiac function in our disease models. Our data suggest ncRNA IGBP1P1 as a potential therapeutic target to improve cardiac function in CVDs.

Quantitative Acetylomics Uncover Acetylation-Mediated Pathway Changes Following Histone Deacetylase Inhibition in Anaplastic Large Cell Lymphoma.

Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1. Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies.

EEF1A1 transcription cofactor gene polymorphism is associated with muscle gene expression and residual feed intake in Nelore cattle.

Cis-acting effects of noncoding variants on gene expression and regulatory molecules constitute a significant factor for phenotypic variation in complex traits. To provide new insights into the impacts of single-nucleotide polymorphisms (SNPs) on transcription factors (TFs) and transcription cofactors (TcoF) coding genes, we carried out a multi-omic analysis to identify cis-regulatory effects of SNPs on these genes' expression in muscle and describe their association with feed efficiency-related traits in Nelore cattle. As a result, we identified one SNP, the rs137256008C > T, predicted to impact the EEF1A1 gene expression (β = 3.02; P-value = 3.51E-03) and the residual feed intake trait (β = -3.47; P-value = 0.02). This SNP was predicted to modify transcription factor sites and overlaps with several QTL for feed efficiency traits. In addition, co-expression network analyses showed that animals containing the T allele of the rs137256008 SNP may be triggering changes in the gene network. Therefore, our analyses reinforce and contribute to a better understanding of the biological mechanisms underlying gene expression control of feed efficiency traits in bovines. The cis-regulatory SNP can be used as biomarker for feed efficiency in Nelore cattle.

P-052: Identification of novel genetic loci for risk of multiple myeloma by functional annotation

<h3>Background</h3> Genetic factors have proven to have an impact on multiple myeloma (MM) susceptibility, with genome-wide association studies (GWAS) discovering 23 loci associated with MM risk. However, much of the heritability of MM remains unexplained. The stringent significance threshold used in GWAS (p<5×10^-8) accounts for the numerous statistical tests being performed but is prone to the risk of false negatives. One strategy for reducing the number of tests is to consider only SNPs with heightened prior probabilities of association, according to meaningful biological criteria. The best candidate SNPs identified with this approach can then be tested in additional MM cases and controls from independent populations. We aimed at surveying the effect of single nucleotide polymorphisms (SNPs), predicted to have a functional role, on MM risk. <h3>Methods</h3> The association study consisted of two GWAS as discovery datasets, namely the InterLymph consortium and the German GWAS and a replication dataset, namely the the International Multiple Myeloma rESEarch (IMMEnSE) consortium, for an overall total of 5442 MM cases and 6174 controls. SNPs were first ranked according to p-value and concordance of the association between the two discovery datasets, then ranked by functional annotation, using bioinformatic tools and databases. We considered the following classes of functional SNPs: missense, synonymous and non-sense SNPs, expression quantitative trait loci (eQTLs), splicing quantitative trait loci (sQTLs),), SNPs in SNPs affecting function of long non-coding RNAs (lncRNA), and SNPs modifying transcription factor binding sites. We prioritized the resulting SNPs for replication in IMMEnSE by p-values for association in InterLymph and German GWAS and by evidence for a functional role. <h3>Results</h3> In the two discovery datasets, 136 SNPs fit the criteria of association with MM risk with p<10^-4 and did not map close to known MM risk loci. After pruning for linkage disequilibrium, four SNPs (rs12038685, rs2664188, rs12652920, rs29794) were chosen for replication in IMMEnSE. Among these, rs2664188 showed to be significantly associated also in the replication dataset (OR=1.30, 95% CI=1.16-1.46, p=0.001). The final meta-analysis including the three datasets, using a random-effect model, confirmed the association (OR=1.18, 95% CI=1.07-1.30, p=0.0007). <h3>Conclusion</h3> The G-allele of rs2664188, that showed a consistent association with increased risk of developing MM in all phases of our analysis is an eQTL, according to GTEx., The G-allele is associated with increased expression of the N4BP2 gene in whole blood. N4BP2 encodes a protein which binds to B-cell leukemia/lymphoma 3 (BCL-3), a well-known proto-oncogene, known to play a role in cell proliferation and apoptosis inhibition in myeloma cell lines.

Single nucleotide polymorphisms in the bovine SLC2A12 and SLC5A1 glucose transporter genes – the effect on gene expression and milk traits of Holstein Friesian cows

In this study, novel single nucleotide polymorphisms (SNPs) were found in the 5′-regulatory regions (promoters) of the bovine glucose transporter (GT) genes SLC2A12 and SLC5A1. These polymorphisms were shown to associate with certain milk production traits in HF cows, including milk yield, milk composition, and somatic cell count. It was shown that the SNP g.-671C > G (NC_037336.1: g.72224078C > G) in the SLC2A12 gene could be an effective marker of cattle production traits and that genotypes CC and CG are associated with the best productivity. The polymorphisms found in the SLC5A1 gene promoter also influenced milk production traits in HF cows, albeit to a lesser extent, and we propose that these polymorphisms could be useful as genetic markers for milk production traits in marker-assisted selection (MAS); however, this must be confirmed on larger populations of cattle. In addition, the presence of polymorphisms within promoter regions appears to affect the expression of GT genes in the cow mammary gland and modify transcription factor (TF) binding capacity.

Basics of Epigenetics and Role of Epigenetics in Diabetic Complications.

The term “Epigenetics” includes mechanisms by which genetic expression is altered without a change in the underlying DNA sequence. The changes caused by epigenetic mechanisms are inheritable and are one way in direction (irreversible) and also explains why there is differences in genetic expressions of monozygotic twins. The epigenetic mechanisms alter the genetic expressions through DNA methylation, posttranslational modifications (PTMs) of histone, and noncoding RNAs. DNA methylation and histone PTMs cause relaxation or condensation of chromatin units. The epigenetic actions of noncoding RNAs such as microRNAs, small nucleolar RNAs, small interfering RNAs, and long noncoding RNAs act by modifying transcription factors or by degrading target messenger RNAs and their translation factors. Various pathologies and environmental factors cause changes in the cellular epigenetic mechanisms and the epigenetic alterations occurring in diabetes mellitus (DM) are reviewed. DM causes hemodynamic changes and metabolic changes like hyperglycemia and dyslipidemia. These changes induce oxidative stress and activate intracellular signaling and kinases in the target cells. Epigenetic alterations cause chromatin remodeling and altered gene expression leading to inflammation, proliferation, atrophy, hypertrophy, etc.; thereby, diabetic complications such as neuropathy, nephropathy, vasculitis result in the corresponding target organ. When these epigenetic alterations persist for a longer period without intervention, the target cells attain “metabolic memory” meaning that these epigenetic mutations cannot be reversed even after attaining normal blood glucose levels. Thus, epigenetics, an insightful and efficient tool in genomic research, has started crawling into the research arena and needs to reach leaps and bounds for the better understanding of health and diseases.

Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.

Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

BackgroundDNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. ResultsThe median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). ConclusionFindings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

Mild iron overload induces TRIP12-mediated degradation of YY1 to trigger hepatic inflammation

Increasing populations are found to bear mild hepatic iron overload (HIO) due to unhealthy lifestyles, metabolic diseases, etc., whether this mild but chronic HIO induces hepatic inflammation is unknown. In the present study, mice receiving a 12-months 0.3% dextran-iron diet show mild HIO with no detectable oxidative damages in the liver but have infiltrated macrophages and increased IL-6, TNFα, AST and ALT since 6-months. The HNF4α/miR-122/CCL2 pathway, identified by our previous studies to induce macrophages infiltration, is initiated by chronic mild HIO. After excluding the role of DNA methylation, a modified transcription factor microarray is applied to find that transcription factor YY1 is responsible for HIO-decreased HNF4α expression. Then the E3 ubiquitin ligase TRIP12 is identified by an immunoprecipitation coupled LC-MS/MS and proved to bind and ubiquitinate YY1, leading to its degradation. The overexpression or silence of YY1 in the liver regulates the HNF4α/miR-122/CCL2 pathway. More importantly, YY1 overexpression alleviates chronic mild HIO induced hepatic inflammatory responses. In conclusion, these results elucidate an oxidative-stress-independent, TRIP12/YY1/HNF4α/miR-122/CCL2 pathway of chronic mild HIO inducing hepatic inflammation, implying that effective measures in addition to antioxidants are needed for individuals at the risk of chronic mild HIO.

Post-Translational Modifications of Transcription Factors Harnessing the Etiology and Pathophysiology in Colonic Diseases.

Defects in mucosal immune balance can lead to colonic diseases such as inflammatory bowel diseases and colorectal cancer. With the advancement of understanding for the immunological and molecular basis of colonic disease, therapies targeting transcription factors have become a potential approach for the treatment of colonic disease. To date, the biomedical significance of unique post-translational modifications on transcription factors has been identified, including phosphorylation, methylation, acetylation, ubiquitination, SUMOylation, and O-GlcNAcylation. This review focuses on our current understanding and the emerging evidence of how post-translational regulations modify transcription factors involved in the etiology and pathophysiology of colonic disease as well as the implications of these findings for new therapeutic approaches in these disorders.

Precision in a rush: Trade-offs between reproducibility and steepness of the hunchback expression pattern.

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene’s ability to read positional information along the embryo’s anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies.

Modulation of transcriptional activity in brain lower grade glioma by alternative splicing

Proteins that modify the activity of transcription factors (TFs) are often called modulators and play a vital role in gene transcriptional regulation. Alternative splicing is a critical step of gene processing, and differentially spliced isoforms may have different functions. Alternative splicing can modulate gene function by adding or removing certain protein domains and thereby influence the activity of a protein. The objective of this study is to investigate the role of alternative splicing in modulating the transcriptional regulation in brain lower grade glioma (LGG), especially transcription factor ELK1, which is closely related to various disorders, including Alzheimer’s disease and Down syndrome. The results showed that changes in the exon inclusion ratio of proteins APP and STK16 are associated with changes in the expression correlation between ELK1 and its targets. In addition, the structural features of the two modulators are strongly associated with the pathological impact of exon inclusion. The results of our analysis suggest that alternatively spliced proteins have different functions in modifying transcription factors and can thereby induce the dysregulation of multiple genes.

Hemorheological alterations in sickle cell anemia and their clinical consequences - The role of genetic modulators.

Sickle cell anemia (SCA) is an autosomal recessive disease caused by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents with high clinical heterogeneity characterized by chronic hemolysis, recurrent episodes of vaso-oclusion and infection. This work aimed to characterize by in silico studies some genetic modulators of severe hemolysis and stroke risk in children with SCA, and understand their consequences at the hemorheological level.Association studies were performed between hemolysis biomarkers as well as the degree of cerebral vasculopathy and the inheritance of several polymorphic regions in genes related with vascular cell adhesion and vascular tonus in pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to investigate the main variant consequences.Variants in vascular adhesion molecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3) gene were significantly associated with higher degree of hemolysis and stroke events. They potentially modify transcription factor binding sites (e.g. VCAM1 rs1409419_T allele may lead to an EVI1 gain) or disturb the corresponding protein structure/function. Our findings emphasize the relevance of genetic variation in modulating the disease severity due to their effect on gene expression or modification of protein biological activities related with sickled erythrocyte/endothelial interactions and consequent hemorheological abnormalities.

Genetically modifying transcription factors to promote CNS axon regeneration.

Genetically modifying transcription factors to promote CNS axon regeneration.

Engineering of synthetic, stress-responsive yeast promoters.

Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.

The Gene Expression Program for the Formation of Wing Cuticle in Drosophila.

The cuticular exoskeleton of insects and other arthropods is a remarkably versatile material with a complex multilayer structure. We made use of the ability to isolate cuticle synthesizing cells in relatively pure form by dissecting pupal wings and we used RNAseq to identify genes expressed during the formation of the adult wing cuticle. We observed dramatic changes in gene expression during cuticle deposition, and combined with transmission electron microscopy, we were able to identify candidate genes for the deposition of the different cuticular layers. Among genes of interest that dramatically change their expression during the cuticle deposition program are ones that encode cuticle proteins, ZP domain proteins, cuticle modifying proteins and transcription factors, as well as genes of unknown function. A striking finding is that mutations in a number of genes that are expressed almost exclusively during the deposition of the envelope (the thin outermost layer that is deposited first) result in gross defects in the procuticle (the thick chitinous layer that is deposited last). An attractive hypothesis to explain this is that the deposition of the different cuticle layers is not independent with the envelope instructing the formation of later layers. Alternatively, some of the genes expressed during the deposition of the envelope could form a platform that is essential for the deposition of all cuticle layers.