cis-DNA Elements Research Articles

Regulon: An overview of plant abiotic stress transcriptional regulatory system and role in transgenic plants.

Population growth is increasing rapidly around the world, in these consequences we need to produce more foods to full fill the demand of increased population. The world is facing global warming due to urbanizations and industrialization and in this concerns plants exposed continuously to abiotic stresses which is a major cause of crop hammering every year. Abiotic stresses consist of Drought, Salt, Heat, Cold, Oxidative and Metal toxicity which damage the crop yield continuously. Drought and salinity stress severally affected in similar manner to plant and the leading cause of reduction in crop yield. Plants respond to various stimuli under abiotic or biotic stress condition and express certain genes either structural or regulatory genes which maintain the plant integrity. The regulatory genes primarily the transcription factors that exert their activity by binding to certain cis DNA elements and consequently either up regulated or down regulate to target expression. These transcription factors are known as masters regulators because its single transcript regulate more than one gene, in this context the regulon word is fascinating more in compass of transcription factors. Progress has been made to better understand about effect of regulons (AREB/ABF, DREB, MYB, and NAC) under abiotic stresses and a number of regulons reported for stress responsive and used as a better transgenic tool of Arabidopsis and Rice.

HyPRP1, A Tomato Multipotent Regulator, Negatively Regulates Tomato Resistance to Sulfur Dioxide Toxicity and Can Also Reduce Abiotic Stress Tolerance of Escherichia coli and Tobacco

Abiotic stresses have led to an extensive decline in global crop production and quality. As one of the abiotic stress factors, sulfur dioxide (SO2) causes severe oxidative damage to plant tissues. Based on our previous study, a tomato hybrid-proline-rich protein 1 (HyPRP1) was found to be involved in abiotic stress and SO2 metabolism, though the gene functions remained largely unknown. In this study, the function analysis of the HyPRP1 gene was extended, and DNA methylation analysis, subcellular localization, and cis-element analysis were performed to investigate the features of this gene. The DNA methylation analysis implied that the HyPRP1 gene was hypermethylated and the methylation density in the leaf differed from that in the flower and fruit. Subcellular localization analysis identified HyPRP1 localized in the cytoplasm and plasma membrane in vivo. The E. coli cells harboring SlHyPRP1 showed reduced salt and drought resistance. In tomato, when SO2 toxicity occurred, the HyPRP1 RNAi knockdown lines accumulated more sulfates and less hydrogen peroxide (H2O2) and showed minimal leaf necrosis and chlorophyll bleaching. In tobacco, the overexpression of HyPRP1 reduced tolerance against salt stresses exerted by NaCl. We conclude that the heterologous expression of tomato HyPRP1 in E. coli and tobacco reduces abiotic stress tolerance and negatively regulates the resistance to sulfur dioxide toxicity by scavenging H2O2 and sulfite in tomato.

The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis.

A key function of photoreceptor signaling is the coordinated regulation of a large number of genes to optimize plant growth and development. The basic helix loop helix (bHLH) transcription factor MYC2 is crucial for regulating gene expression in Arabidopsis thaliana during development in blue light. Here we demonstrate that blue light induces the SUMOylation of MYC2. Non-SUMOylatable MYC2 is less effective in suppressing blue light-mediated photomorphogenesis than wild-type (WT) MYC2. MYC2 interacts physically with the SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2. Blue light exposure promotes the degradation of SPF1 and SPF2 and enhances the SUMOylation of MYC2. Phenotypic analysis revealed that SPF1/SPF2 function redundantly as positive regulators of blue light-mediated photomorphogenesis. Our data demonstrate that SUMO conjugation does not affect the dimerization of MYC transcription factors but modulates the interaction of MYC2 with its cognate DNA cis-element and with the ubiquitin ligase Plant U-box 10 (PUB10). Finally, we show that non-SUMOylatable MYC2 is less stable and interacts more strongly with PUB10 than the WT. Taken together, we conclude that SUMO functions as a counterpoint to the ubiquitin-mediated degradation of MYC2, thereby enhancing its function in blue light signaling.

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless.

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. In this study, we utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. Our results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. We further show that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, our study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1.

In Vivo Function of Flow-Responsive Cis-DNA Elements of eNOS Gene: A Role for Chromatin-Based Mechanisms.

eNOS (endothelial nitric oxide synthase) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress. Disturbed flow with lower average shear stress decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains 2 distinct flow-responsive cis-DNA elements in the promoter, the shear stress response element (SSRE) and the KLF (Krüppel-like factor) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis-DNA elements remains unknown. Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro. DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Wild type mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). It is surprising that the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis-element necessary for eNOS transcription in vivo. The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wild type mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo. We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic shear stress in vivo. Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Identification of a 22bp DNA cis Element that Plays a Critical Role in Colony Stimulating Factor 1-Dependent Transcriptional Activation of the SPHK1 Gene.

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Colony Stimulating Factor 1 (CSF1) induces expression of the rate limiting enzyme required for S1P synthesis, sphingosine kinase 1 (SPHK1) in bone in vivo, and in osteoclasts in vitro. To study the mechanism of CSF1-induced SPHK1 gene expression, a 2608bp fragment of the murine SPHK1 gene (- 2497 to + 111bp relative to the transcription start site) was cloned and transfected into pZen cells (murine fibroblasts engineered to express c-fms). SPHK1 promoter activity was assessed using a dual-luciferase reporter assay system. By analyzing a series of 5'-deletions, a CSF1-responsive region was identified in the region - 1250 to - 1016bp. To define putative DNA binding site(s) in this fragment, two biotin-labeled fragments that completely overlapped this region were generated, one 163bp in length (- 1301 to - 1139) and one 169bp in length (- 1157 to - 989). EMSAs revealed the 163bp fragment as the target for protein binding. Using EMSAs, the nuclear protein binding region was further narrowed to an 85bp fragment, (- 1223 to - 1139). Using a series of unlabeled DNA sequences as "cold competitors" in EMSAs, a 22bp sequence is identified as the smallest fragment that could successfully compete away protein binding. The same 22bp sequence also competed DNA binding in EMSAs using nuclear protein isolated from primary murine osteoclasts. A full-length wild-type SPHK1 promoter and an SPHK1 promoter in which the ATGGGGG motif was mutated were subsequently expressed in pZen cells. Mutating this ATGGGGG motif nearly completely abrogated the ability of CSF1 to activate the promoter. Although two transcription factors, KLF6 and Sp1 have been reported to bind to this sequence, supershift EMSAs failed to detect either among the proteins bound to the 85bp DNA fragment.

Brain-specific homeobox Bsx specifies identity of pineal gland between serially homologous photoreceptive organs in zebrafish

The pineal gland functioning as a photoreceptive organ in non-mammalian species is a serial homolog of the retina. Here we found that Brain-specific homeobox (Bsx) is a key regulator conferring individuality on the pineal gland between the two serially homologous photoreceptive organs in zebrafish. Bsx knock-down impaired the pineal development with reduced expression of exorh, the pineal-specific gene responsible for the photoreception, whereas it induced ectopic expression of rho, a retina-specific gene, in the pineal gland. Bsx remarkably transactivated the exorh promoter in combination with Otx5, but not with Crx, through its binding to distinct subtypes of PIRE, a DNA cis-element driving Crx/Otx-dependent pineal-specific gene expression. These results demonstrate that the identity of pineal photoreceptive neurons is determined by the combinatorial code of Bsx and Otx5, the former confers the pineal specificity at the tissue level and the latter determines the photoreceptor specificity at the cellular level.

Functional D-box sequences reset the circadian clock and drive mRNA rhythms

The circadian clock drives gene expression rhythms, leading to daily changes in physiology and behavior. In mammals, Albumin D-site-Binding Protein (DBP) rhythmically activates transcription of various genes through a DNA cis-element, D-box. The DBP-dependent transactivation is repressed by competitive binding of E4BP4 to the D-box. Despite the elaborate regulation, physiological roles of the D-box in the circadian clockwork are still elusive. Here we identified 1490 genomic regions recognized commonly by DBP and E4BP4 in the mouse liver. We comprehensively defined functional D-box sequences using an improved bioinformatics method, MOCCS2. In RNA-Seq analysis of E4bp4-knockout and wild type liver, we showed the importance of E4BP4-mediated circadian repression in gene expression rhythms. In addition to the circadian control, we found that environmental stimuli caused acute induction of E4BP4 protein, evoking phase-dependent phase shifts of cellular circadian rhythms and resetting the clock. Collectively, D-box-mediated transcriptional regulation plays pivotal roles in input and output in the circadian clock system.

Regulation of GLI1 by cis DNA elements and epigenetic marks

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

Overexpression of Soybean Transcription Factors GmDof4 and GmDof11 Significantly Increase the Oleic Acid Content in Seed of Brassica napus L.

Rapeseed (Brassica napus L.) with substantial lipid and oleic acid content is of great interest to rapeseed breeders. Overexpression of Glycine max transcription factors Dof4 and Dof11 increased lipid accumulation in Arabidopsis and microalgae, in addition to modifying the quantity of certain fatty acid components. Here, we report the involvement of GmDof4 and GmDof11 in regulating fatty acid composition in rapeseeds. Overexpression of GmDof4 and GmDof11 in rapeseed increased oleic acid content and reduced linoleic acid and linolenic acid. Both qPCR and the yeast one-hybrid assay indicated that GmDof4 activated the expression of FAB2 by directly binding to the cis-DNA element on its promoters, while GmDof11 directly inhibited the expression of FAD2. Thus, GmDof4 and GmDof11 might modify the oleic acid content in rapeseed by directly regulating the genes that are associated with fatty acid biosynthesis.

In vivo Function of Flow-Responsive cis-DNA Elements of the Endothelial Nitric Oxide Synthase Gene: a Role for Chromatin-based Mechanisms

In vivo Function of Flow-Responsive cis-DNA Elements of the Endothelial Nitric Oxide Synthase Gene: a Role for Chromatin-based Mechanisms

Expression and deletion analyses of cspE encoding cold-shock protein E in Acinetobacter oleivorans DR1

Six genes encoding cold-shock-like proteins, including cspE, are contained within the genome of Acinetobacter oleivorans DR1. All six genes are similar in size as well as amino acid identity, but appear to be differentially regulated under stressful conditions. Four of these genes (cspA, cspB, cspC and cspE) were functionally important during cold shock because of their gradual upregulation during a temperature decrease under our assay conditions. cspE also showed higher expression during alkane degradation and antibiotic exposure. The transcriptional start site of the cspE gene was determined using 5′ rapid amplification of complementary DNA ends. Next, promoter analysis using numerous constructed gfp reporter strains containing deleted fragments of cspE upstream regions identified possible 5′ untranslated region (UTR) cis-DNA elements that could be involved in modulating cspE expression. Deletion of cspE led to a growth defect and enhanced biofilm formation, but only at a low temperature. Collectively, our findings show the importance of CspE during cold shock, dynamic regulation of cspE expression under various stressful conditions and a possible 5′-UTR cis-DNA element for regulation of cspE expression. These data provide molecular insight into cspE gene expression during cold-shock adaptation in soil bacteria.

The STENOFOLIA gene from Medicago alters leaf width, flowering time and chlorophyll content in transgenic wheat.

SummaryMolecular genetic analyses revealed that the WUSCHEL‐related homeobox (WOX) gene superfamily regulates several programs in plant development. Many different mechanisms are reported to underlie these alterations. The WOX family member STENOFOLIA (STF) is involved in leaf expansion in the eudicot Medicago truncutula. Here, we report that when this gene was ectopically expressed in a locally adapted hard red winter wheat cultivar (Triticum aestivum), the transgenic plants showed not only widened leaves but also accelerated flowering and increased chlorophyll content. These desirable traits were stably inherited in the progeny plants. STF binds to wheat genes that have the (GA)n/(CT)n DNA cis element, regardless of sequences flanking the DNA repeats, suggesting a mechanism for its pleiotropic effects. However, the amino acids between position 91 and 262 in the STF protein that were found to bind with the (GA)n motif have no conserved domain with any other GAGA‐binding proteins in animals or plants. We also found that STF interacted with a variety of proteins in wheat in yeast 2 hybrid assays. We conclude that the eudicot STF gene binds to (GA)n/(CT)n DNA elements and can be used to regulate leaf width, flowering time and chlorophyll content in monocot wheat.

The LIM protein complex establishes a retinal circuitry of visual adaptation by regulating Pax6 α-enhancer activity.

The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of Paired-homeobox 6 (PAX6) is regulated by multiple cis-DNA elements including the intronic α-enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the α-enhancer in the post-natal mouse retina. Tgfb1i1-/- mice show elevated α-enhancer activity leading to overproduction of Pax6ΔPD isoform that supports the GABAergic amacrine cell fate maintenance. Consequently, the Tgfb1i1-/- mouse retinas show a sustained light response, which becomes more transient in mice with the auto-stimulation-defective Pax6ΔPBS/ΔPBS mutation. Together, we show the antagonistic regulation of the α-enhancer activity by Pax6 and the LIM protein complex is necessary for the establishment of an inner retinal circuitry, which controls visual adaptation.

Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.

Gene expression requires the coordination of trans-acting factors and cis-DNA elements to initiate transcription. Here we present a coordinated approach that combines cis-acting element engineering with mutant trans-acting factors to engineer yeast promoters. Specifically, we first construct a hybrid promoter based on the ARO9 upstream region that exhibits high constitutive and inducible expression with respect to exogenous tryptophan. Next, we perform protein engineering to identify a mutant Aro80p that affords both high constitutive expression while retaining inducible traits. We then use this mutant trans-acting factor to drive expression and generate ultra-strong promoters with transcriptional output roughly 2 fold higher than TDH3 (GPD), one of the strongest promoters to-date. Finally, we used this element to construct a modular expression system capable of staged outputs resulting in a system with nearly 6-fold, 12-fold and 15-fold expression relative to the off-state. This work further highlights the potential of using endogenous transcription factors (including mutant factors) along with hybrid promoters to expand the yeast synthetic biology toolbox.

T-2 toxin induces the expression of porcine CYP3A22 via the upregulation of the transcription factor, NF-Y

BackgroundT-2 toxin is one of the major pollutants in crops and feedstuffs. CYP3A22, one of hCYP3A4 homologs, detoxifies T-2 toxin in pigs. We investigated the mechanisms of expression activation of CYP3A22 under basal and induced conditions. MethodsBased on MatInspector analysis, several mutations in the CYP3A22 promoter were assayed by dual luciferase reporter to identify the function of cis elements in the region. EMSA experiments were used to assess the binding of transcription factors to the cis elements. The mRNA and protein levels of CYP3A22 and the transcription factors were measured by RT-qPCR and Western blot. The enhancement of NF-Y binding to the CYP3A22 promoter was assayed by ChIP. ResultsAs predicted, two cis DNA elements in the CYP3A22 promoter, a CCAAT box and GC box, were confirmed to be crucial in the activation of CYP3A22 transcription. These two DNA motifs recruited two transcription factors, NF-Y and Sp1, which are involved in the activation of the basal transcription of CYP3A22. More interestingly, CYP3A22 expression was induced in porcine primary hepatocytes by the treatment with 0.1μg/mL T-2 toxin. This induction of transcription by T-2 toxin was dominantly regulated by the binding of NF-Y to the CCAAT box, rather than GC box, which recruits Sp1 and functions only in the constitutive expression of CYP3A22. ConclusionsOur study reveals the regulatory mechanisms of both basal and inducible transactivation of CYP3A22 in pigs. In particular, we identified that the mechanism by which T-2 toxin induces CYP3A22 expression is mediated by the upregulation of NF-YA. General significanceAlthough porcine CYP3A22 is homologous to hCYP3A4, the regulation of basal and induced expression of CYP3A22 occurred via distinct mechanisms. This may account for the variety of CYP3A expression in animals and humans.

Intronic regulation of human GLI1 DNA by cis DNA elements and epigenetic marks

In humans, a large cancer burden is associated with dysregulation of the Hedgehog/Patched/GLI (HH/PCTH/GLI) pathway. The canonical Hedgehog signal (including Sonic Hedgehog [SHH]) is mediated by the GLI family of transcription factors (GLI1, GLI2 and GLI3). GLI1 plays an important role in normal development and differentiation and is a human oncogene that is believed to be active in up to one‐third of all cancers. GLI1 gene targets sustain proliferation, inhibit apoptosis, and promote angiogenesis and tumor cell migration. Wild‐type p53 competes with GLI1 for the co‐activator TAF9, inhibiting GLI1's oncogenic activity. GLI1 and GLI2 form a positive‐feedback loop that drives their expression. Mechanisms that regulate this positive‐feedback loop remain unknown. We identified 6 GLI binding sites (GBS) in the large first intron of the human GLI1 gene that are highly conserved among mammals and are associated with histone H3K27Ac marks and DNase hypersensitivity clusters. GLI1 binds all of the GBS and GLI2 binds 2 of them, shown using gel mobility shift assays. Elimination of some of the GBS attenuates transcriptional activation. Removal of all GBS eliminates reporter gene activation. Preliminary evidence suggests GLI1 multimerizes and can bind the pioneer protein CBP. ChIP qPCR shows SHH signal dependent (Smoothened agonist) occupancy of the 1st intron near the GBS by GLI2, and increased occupancy by the histones H3K4me3 and H3K27Ac and the histone bromodomain reader protein BRD4. BRD4 can be driven off the intron and transcription start site region with a small inhibitory molecule, I‐BET151, providing a possible route to disrupting GLI1 expression. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 could auto‐regulate GLI1 expression at GBS in the 1st intron of GLI1 through protein‐protein interactions involving complexes of the transcription factors, histone variants, and reader proteins. Given the widespread involvement of GLI1 and GLI2 in human cancers including medulloblastoma and rhabdomyosarcoma, it is important to try and understand key elements of the regulation of their expression.Support or Funding InformationSupported in part by PHS grant ES1054, Illinois Excellence in Academic Medicine award, Illinois Regenerative Medicine Institute and the Eisenberg Foundation for Charities.

Genetic and Epigenetic Regulation of PD-1 Expression.

The inhibitory immune receptor programmed cell death-1 (PD-1) is intricately regulated. In T cells, PD-1 is expressed in response to most immune challenges, but it is rapidly downregulated in acute settings, allowing for normal immune responses. On chronically stimulated Ag-specific T cells, PD-1 expression remains high, leading to an impaired response to stimuli. Ab blockade of PD-1 interactions during chronic Ag settings partially restores immune function and is now used clinically to treat a variety of devastating cancers. Understanding the regulation of PD-1 expression may be useful for developing novel immune-based therapies. In this review, the molecular mechanisms that drive dynamic PD-1 expression during acute and chronic antigenic stimuli are discussed. An array of cis-DNA elements, transcription factors, and epigenetic components, including DNA methylation and histone modifications, control PD-1 expression. The interplay between these regulators fine-tunes PD-1 expression in different inflammatory environments and across numerous cell types to modulate immune responses.

The SOX11 transcription factor is a critical regulator of basal-like breast cancer growth, invasion, and basal-like gene expression.

Basal-like breast cancers (BLBCs) are aggressive breast cancers associated with poor survival. Defining the key drivers of BLBC growth will allow identification of molecules for targeted therapy. In this study, we performed a primary screen integrating multiple assays that compare transcription factor expression and activity in BLBC and non-BLBC at the RNA, DNA, and protein levels. This integrated screen identified 33 transcription factors that were elevated in BLBC in multiple assays comparing mRNA expression, DNA cis-element sequences, or protein DNA-binding activity. In a secondary screen to identify transcription factors critical for BLBC cell growth, 8 of the 33 candidate transcription factors (TFs) were found to be necessary for growth in at least two of three BLBC cell lines. Of these 8 transcription factors, SOX11 was the only transcription factor required for BLBC growth, but not for growth of non-BLBC cells. Our studies demonstrate that SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer.

Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element

Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.