The mesodermal and myogenic specification of hESCs depend on ZEB1 and are inhibited by ZEB2.

Human embryonic stem cells (hESCs) can differentiate into any cell lineage. Here, we report that ZEB1 and ZEB2 promote and inhibit mesodermal-to-myogenic specification of hESCs, respectively. Knockdown and/or overexpression experiments of ZEB1, ZEB2, or PAX7 in hESCs indicate that ZEB1 is required for hESC Nodal/Activin-mediated mesodermal specification and PAX7+ human myogenic progenitor (hMuP) generation, while ZEB2 inhibits these processes. ZEB1 downregulation induces neural markers, while ZEB2 downregulation induces mesodermal/myogenic markers. Mechanistically, ZEB1 binds to and transcriptionally activates the PAX7 promoter, while ZEB2 binds to and activates the promoter of the neural OTX2 marker. Transplanting ZEB1 or ZEB2 knocked down hMuPs into the muscles of a muscular dystrophy mouse model, showing that hMuP engraftment and generation of dystrophin-positive myofibers depend on ZEB1 and are inhibited by ZEB2. The mouse model results suggest that ZEB1 expression and/or downregulating ZEB2 in hESCs may also enhance hESC regenerative capacity for human muscular dystrophy therapy.

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

A causal inference and Bayesian optimisation framework for modelling multi-trait relationships-Proof-of-concept using Brassica napus seed yield under controlled conditions.

The improvement of crop yield is a major breeding target and there is a long history of research that has focussed on unravelling the mechanisms and processes that contribute to yield. Quantitative prediction of the interplay between morphological traits, and the effects of these trait-trait relationships on seed production remains, however, a challenge. Consequently, the extent to which crop varieties optimise their morphology for a given environment is largely unknown. This work presents a new combination of existing methodologies by framing crop breeding as an optimisation problem and evaluates the extent to which existing varieties exhibit optimal morphologies under the test conditions. In this proof-of-concept study using spring and winter oilseed rape plants grown under greenhouse conditions, we employ causal inference to model the hierarchically structured effects of 27 morphological yield traits on each other. We perform Bayesian optimisation of seed yield, to identify and quantify the morphologies of ideotype plants, which are expected to be higher yielding than the varieties in the studied panels. Under the tested growth conditions, we find that existing spring varieties occupy the optimal regions of trait-space, but that potentially high yielding strategies are unexplored in extant winter varieties. The same approach can be used to evaluate trait (morphology) space for any environment.

Automated extraction of pod phenotype data from micro-computed tomography.

Introduction: Plant image datasets have the potential to greatly improve our understanding of the phenotypic response of plants to environmental and genetic factors. However, manual data extraction from such datasets are known to be time-consuming and resource intensive. Therefore, the development of efficient and reliable machine learning methods for extracting phenotype data from plant imagery is crucial. Methods: In this paper, a current gold standard computed vision method for detecting and segmenting objects in three-dimensional imagery (StartDist-3D) is applied to X-ray micro-computed tomography scans of oilseed rape (Brassica napus) mature pods. Results: With a relatively minimal training effort, this fine-tuned StarDist-3D model accurately detected (Validation F1-score = 96.3%,Testing F1-score = 99.3%) and predicted the shape (mean matched score = 90%) of seeds. Discussion: This method then allowed rapid extraction of data on the number, size, shape, seed spacing and seed location in specific valves that can be integrated into models of plant development or crop yield. Additionally, the fine-tuned StarDist-3D provides an efficient way to create a dataset of segmented images of individual seeds that could be used to further explore the factors affecting seed development, abortion and maturation synchrony within the pod. There is also potential for the fine-tuned Stardist-3D method to be applied to imagery of seeds from other plant species, as well as imagery of similarly shaped plant structures such as beans or wheat grains, provided the structures targeted for detection and segmentation can be described as star-convex polygons.

Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients.

Inherited retinal dystrophies comprise a broad group of genetic eye diseases without effective treatment. Among them, Stargardt disease is the second most prevalent pathology. This pathology triggers progressive retinal degeneration and vision loss in children and adults. In recent years, the evolution of several genome editing technologies, such as the CRISPR-Cas9 system, has revolutionized disease modeling and personalized medicine. Human induced pluripotent stem cells also provide a valuable tool for in vitro disease studies and therapeutic applications. Here, we show precise correction of two ABCA4 pathogenic variants in human induced pluripotent stem cells from two unrelated patients affected with Stargardt disease. Gene editing was achieved with no detectable off-target genomic alterations, demonstrating efficient ABCA4 gene correction without deleterious effects. These results will contribute to the development of emerging gene and cell therapies for inherited retinal dystrophies.

High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs.

Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.

A new mutation in the PDE6C gene in achromatopsia.

PURPOSE: To report a case of achromatopsia with a new mutation in the PDE6C gene which has not been previously described. METHODS: Case report. PATIENTS: A single patient. RESULTS: A 35-year-old woman with poor vision and impaired color vision. Fundus examination of both eyes (OU) revealed small optic discs. Optical coherence tomography (OCT) showed photoreceptor segment defects and a disruption of the ellipsoid layer in the foveal region, with intact overlying outer limiting membrane and underlying RPE bands. The electroretinogram (ERG) showed scotopic responses: DA 0.01: normal amplitude, b-wave latency at upper limit of normal / slightly increased. DA 3 and DA 10: slightly increased b-wave latency, asymmetry in b amplitude, being lower in the left eye. Oscillatory potentials: no responses are obtained. Photopic responses: LA-3: greatly increased latencies, decreased amplitude. Subsequently, a case of incomplete achromatopsia was suspected. Therefore, a genetic study was carried out showing the homozygous presence of the undescribed pathogenic variant c.660_661del (p.Ser221Tyrfs * 15) in exon 3 of the PDE6C gene. CONCLUSION: Achromatopsia is an autosomal-recessive genetic disease characterized by decreased visual acuity, color blindness, photophobia, and nystagmus. Due to the variability of genetic mutations in achromatopsia, genetic characterization is mandatory in order to improve the efficiency in molecular diagnosis. This data may be useful in future therapeutic strategies. We present a previously undescribed mutation in the PDE6C gene in a patient with incomplete achromatopsia.

Uncovering Trait Associations Resulting in Maximal Seed Yield in Winter and Spring Oilseed Rape.

Seed yield is a complex trait for many crop species including oilseed rape (OSR) (Brassica napus), the second most important oilseed crop worldwide. Studies have focused on the contribution of distinct factors in seed yield such as environmental cues, agronomical practices, growth conditions, or specific phenotypic traits at the whole plant level, such as number of pods in a plant. However, how female reproductive traits contribute to whole plant level traits, and hence to seed yield, has been largely ignored. Here, we describe the combined contribution of 33 phenotypic traits within a B. napus diversity set population and their trade-offs at the whole plant and organ level, along with their interaction with plant level traits. Our results revealed that both Winter OSR (WOSR) and Spring OSR (SOSR); the two more economically important OSR groups in terms of oil production; share a common dominant reproductive strategy for seed yield. In this strategy, the main inflorescence is the principal source of seed yield, producing a good number of ovules, a large number of long pods with a concomitantly high number of seeds per pod. Moreover, we observed that WOSR opted for additional reproductive strategies than SOSR, presenting more plasticity to maximise seed yield. Overall, we conclude that OSR adopts a key strategy to ensure maximal seed yield and propose an ideal ideotype highlighting crucial phenotypic traits that could be potential targets for breeding.

Big data from small tissues: extraction of high-quality RNA for RNA-sequencing from different oilseed Brassica seed tissues during seed development.

BACKGROUND: Obtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptomes during seed development. RESULTS: Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and ovary wall tissue were also collected from pre-fertilized buds. Subsequent to testing several RNA extraction methods, modifications applied to E.Z.N.A. Plant RNA and Picopure RNA Isolation kit extraction methods resulted in RNA with high yield and quality. Furthermore, the use of polyvinylpolypyrrolidone for seed coats and endosperm at green stages resulted in high-quality RNA. As a result of the introduced modifications to established RNA extraction methods, the RNA from all the above-mentioned tissues presented clear 28S and 18S bands and high RIN values, ranging from 7.0 to 10.0. The protocols reported in this study are not only suitable for different and challenging seed tissue types, but also enable the extraction of high-quality RNA using only 2 to 3 mg of starting tissue. CONCLUSIONS: Here, we present efficient, reproducible and reliable high-quality RNA extraction methods for diverse oilseed Brassica spp reproductive tissue types including pre-fertilization and developing seed tissues for diploid and polyploid species. The high-quality RNA obtained is suitable for RNA-Sequencing and subsequent gene expression analysis.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells.

OBJECTIVE: Chronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC. DESIGN: The study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination. RESULTS: ZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts, Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1ß). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing of Zeb1-deficient and Mpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1ß production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair. CONCLUSIONS: ZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

ZEB1 protects skeletal muscle from damage and is required for its regeneration.

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.

Regulation of muscle atrophy-related genes by the opposing transcriptional activities of ZEB1/CtBP and FOXO3.

Multiple physiopathological and clinical conditions trigger skeletal muscle atrophy through the induction of a group of proteins (atrogenes) that includes components of the ubiquitin-proteasome and autophagy-lysosomal systems. Atrogenes are induced by FOXO transcription factors, but their regulation is still not fully understood. Here, we showed that the transcription factor ZEB1, best known for promoting tumor progression, inhibits muscle atrophy and atrogene expression by antagonizing FOXO3-mediated induction of atrogenes. Compared to wild-type counterparts, hindlimb immobilization in Zeb1-deficient mice resulted in enhanced muscle atrophy and higher expression of a number of atrogenes, including Atrogin-1/Fbxo32, MuRF1/Trim63, Ctsl, 4ebp1, Gabarapl1, Psma1 and Nrf2. Likewise, in the C2C12 myogenic cell model, ZEB1 knockdown augmented both myotube diameter reduction and atrogene upregulation in response to nutrient deprivation. Mechanistically, ZEB1 directly represses in vitro and in vivo Fbxo32 and Trim63 promoter transcription in a stage-dependent manner and in a reverse pattern with MYOD1. ZEB1 bound to the Fbxo32 promoter in undifferentiated myoblasts and atrophic myotubes, but not in non-atrophic myotubes, where it is displaced by MYOD1. ZEB1 repressed both promoters through CtBP-mediated inhibition of FOXO3 transcriptional activity. These results set ZEB1 as a new target in therapeutic approaches to clinical conditions causing muscle mass loss.

Epigenetically distinct sister chromatids and asymmetric generation of tumor initiating cells.

Cancer stem cells (CSC) are thought to be an important source of cancer cells in tumors of different origins. Mounting evidence suggests they are generated reversibly from existing cancer cells, and supply new cancer cells during tumor progression and following therapy. Elegant lineage mapping stud(ies are identifying progenitors, and in some cases differentiated cells, as targets of transformation in a variety of tumors. Recent evidence suggests resulting tumor initiating cells (TIC) might be distinct from CSC. Molecular pathways leading from cells of tumor origin to precancerous lesions and cancer cells are only beginning to be unraveled. We review a pathway where asymmetric division of precancerous cells generates TIC in a K-Ras-initiated model of lung cancer. And, we compare unexpected steps in this asymmetric division to those evident in well-studied stem cell models.

Transcriptional Regulation of Vitamin E Biosynthesis during Germination of Dwarf Fan Palm Seeds.

Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

Mitotic polarization of transcription factors during asymmetric division establishes fate of forming cancer cells.

A model of K-Ras-initiated lung cancer was used to follow the transition of precancerous adenoma to adenocarcinoma. In hypoxic, Tgf-ß1-rich interiors of adenomas, we show that adenoma cells divide asymmetrically to produce cancer-generating cells highlighted by epithelial mesenchymal transition and a CD44/Zeb1 loop. In these cells, Zeb1 represses the Smad inhibitor Zeb2/Sip1, causing Pten loss and launching Tgf-ß1 signaling that drives nuclear translocation of Yap1. Surprisingly, the nuclear polarization of transcription factors during mitosis establishes parent and daughter fates prior to cytokinesis in sequential asymmetric divisions that generate cancer cells from precancerous lesions. Mutation or knockdown of Zeb1 in the lung blocked the production of CD44hi, Zeb1hi cancer-generating cells from adenoma cells. A CD44/Zeb1 loop then initiates two-step transition of precancerous cells to cancer cells via a stable intermediate population of cancer-generating cells. We show these initial cancer-generating cells are independent of cancer stem cells generated in tumors by p53-regulated reprogramming of existing cancer cells.

ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1.

OBJECTIVE: Understand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition. DESIGN: Expression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including colorectal carcinomas (CRCs). The study uses survival analysis, in vivo mouse transgenic and xenograft models, gene expression arrays, immunostaining and gene and protein regulation assays. RESULTS: The poorer survival determined by ZEB1 in CRCs depended on simultaneous high levels of the Wnt antagonist DKK1, whose expression was transcriptionally activated by ZEB1. In cancer cells with mutant TP53, ZEB1 blocked the formation of senescence-associated heterochromatin foci at the onset of senescence by triggering a new regulatory cascade that involves the subsequent activation of DKK1, mutant p53, Mdm2 and CtBP to ultimately repress macroH2A1 (H2AFY). In a transgenic mouse model of colon cancer, partial downregulation of Zeb1 was sufficient to induce H2afy and to trigger in vivo tumour senescence, thus resulting in reduced tumour load and improved survival. The capacity of ZEB1 to induce tumourigenesis in a xenograft mouse model requires the repression of H2AFY by ZEB1. Lastly, the worst survival effect of ZEB1 in patients with CRC ultimately depends on low expression of H2AFY and of senescence-associated genes. CONCLUSIONS: The tumourigenic capacity of ZEB1 depends on its inhibition of cancer cell senescence through the activation of a herein identified new molecular pathway. These results set ZEB1 as a potential target in therapeutic strategies aimed at inducing senescence.

Enhanced tocopherol levels during early germination events in Chamaerops humilis var. humilis seeds.

Most angiosperms accumulate vitamin E in the form of tocopherols in seeds, exerting a protective antioxidant role. However, several palm trees principally accumulate tocotrienols, rather than tocopherols, in seeds, as it occurs in other monocots. To unravel the protective role of either tocopherols or tocotrienols against lipid peroxidation during seed germination in Chamaerops humilis var. humilis; seed viability, natural and induced germination capacity, seed water content, malondialdehyde levels (as an indicator of the extent of lipid peroxidation) and vitamin E levels (including both tocopherols and tocotrienols) were examined at various germination phases in a simulated, natural seed bank. At the very early stages of germination (operculum removal), malondialdehyde levels increased 2.8-fold, to decrease later up to 74%, thus indicating a transient lipid peroxidation at early stages of germination. Tocopherol levels were absent in quiescent seeds and did not increase during operculum removal, but increased later presumably dampening malondialdehyde accumulation. Thereafter, tocopherols continued increasing, while lipid peroxidation levels decreased. By contrast, tocotrienols levels remained constant or even decreased as germination progressed, showing no correlation with lipid peroxidation levels. We hypothesize that despite their high tocotrienol content, seeds synthesize tocopherols during germination to protect lipids from peroxidation events.

Perennially young: seed production and quality in controlled and natural populations of Cistus albidus reveal compensatory mechanisms that prevent senescence in terms of seed yield and viability.

The question of whether or not perennial plants senesce at the organism level remains unresolved. The aim of this study was to unravel whether or not plant age can influence the production and composition of seeds. Flower and seed production was examined in 3-, 8-, and 13-year-old Cistus albidus plants growing in experimental plots corresponding to the F2, F1, and F0 generations of the same population. Furthermore, the phytohormone, fatty acid, and vitamin E content of the seeds was evaluated, and their viability was examined. Whether or not age-related differences in seed quality were observed in a natural population in the Montserrat Mountains (NE Spain) was also tested. The results indicate that under controlled conditions, the oldest plants not only produced fewer flowers, but also had higher rates of embryo abortion in mature seeds. However, germination capacity was not negatively affected by plant ageing. Seeds of the oldest plants contained significantly higher salicylic acid, jasmonic acid, and vitamin E levels compared with those from younger plants. Despite vigour (in terms of plant growth) being severely reduced due to harsh environmental conditions in the natural population, the oldest individuals produced seeds with no decline in viability. Seed biomass was instead positively correlated with seed viability. In conclusion, increased plant size may explain the loss of seed viability in the experimental field, but older smaller individuals in natural populations can escape senescence in terms of seed viability loss.

Vitamin E analyses in seeds reveal a dominant presence of tocotrienols over tocopherols in the Arecaceae family.

Tocopherols are thought to prevent oxidative damage during seed quiescence and dormancy in all angiosperms. However, several monocot species accumulate tocotrienols in seeds and their role remains elusive. Here, we aimed to unravel the distribution of tocopherols and tocotrienols in seeds of the Arecaceae family, to examine possible trends of vitamin E accumulation within different clades of the same family. We examined the tocopherol and tocotrienol content in seeds of 84 species. Furthermore, we evaluated the vitamin E composition of the seed coat, endosperm and embryo of seeds from 6 species, to determine possible tissue-specific functions of particular vitamin E forms. While seeds of 98.8% (83 out of 84) of the species accumulated tocotrienols, only 58.3% (49 out of 84) accumulated tocopherols. The presence of tocopherols did not follow a clear evolutionary trend, and appeared randomly in some clades only. In addition, the tissue-specific location of vitamin E in seeds revealed that the embryo contains mostly α-tocopherol (in seed tocopherol-accumulating species) or α-tocotrienol (in seed tocopherol-deficient species). However, some species such as Socratea exorrhiza mostly accumulate ß-tocotrienol, and Parajubaea torallyi accumulates a mixture of tocopherols and tocotrienols in the embryo. This suggests that tocotrienols can play a similar protective role to that exerted by tocopherols in seeds, at least in some species of the Arecaceae family. We conclude that tocotrienol, rather than tocopherol, accumulation is a conserved trait in seeds of the Arecaceae family.

ZEB1 Promotes invasiveness of colorectal carcinoma cells through the opposing regulation of uPA and PAI-1.

PURPOSE: Carcinoma cells enhance their invasive capacity through dedifferentiation and dissolution of intercellular adhesions. A key activator of this process is the ZEB1 transcription factor, which is induced in invading cancer cells by canonical Wnt signaling (ß-catenin/TCF4). Tumor invasiveness also entails proteolytic remodeling of the peritumoral stroma. This study aimed to investigate the potential regulation by ZEB1 of the plasminogen proteolytic system constituted by the urokinase plasminogen activator (uPA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). EXPERIMENTAL DESIGN: Through multiple experimental approaches, colorectal carcinoma (CRC) cell lines and samples from human primary CRC and ZEB1 (-/-) mice were used to examine ZEB1-mediated regulation of uPA and PAI-1 at the protein, mRNA, and transcriptional level. RESULTS: ZEB1 regulates uPA and PAI-1 in opposite directions: induces uPA and inhibits PAI-1. In vivo expression of uPA depends on ZEB1 as it is severely reduced in the developing intestine of ZEB1 null (-/-) mice. Optimal induction of uPA by Wnt signaling requires ZEB1 expression. ZEB1 binds to the uPA promoter and activates its transcription through a mechanism implicating the histone acetyltransferase p300. In contrast, inhibition of PAI-1 by ZEB1 does not involve transcriptional repression but rather downregulation of mRNA stability. ZEB1-mediated tumor cell migration and invasion depend on its induction of uPA. ZEB1 coexpresses with uPA in cancer cells at the invasive front of CRCs. CONCLUSIONS: ZEB1 promotes tumor invasiveness not only via induction in cancer cells of a motile dedifferentiated phenotype but also by differential regulation of genes involved in stroma remodeling.