Embryonic Extracts Research Articles

Exogenous maize grain embryonic extract enriched with some biostimulants confers drought stress tolerance by promoting tomato's growth, photosynthetic efficiency, antioxidant defense, and yield and quality.

Exogenous maize grain embryonic extract enriched with some biostimulants confers drought stress tolerance by promoting tomato's growth, photosynthetic efficiency, antioxidant defense, and yield and quality.

The Effect of Cynometra cauliflora Linn. Fruit Extract-Induced Congenital Anomalies on Chick Embryos

Background: Cynometra cauliflora is a traditional folk medicine that is used to treat a variety of unpleasant symptoms without adverse effects. Previous studies have shown that C. cauliflora fruit relieving morning sickness and nausea in pregnant women. However, no research has been performed on the effect of C. cauliflora fruit extract on birth anomalies. Objective: In this study, we aim to investigate the effects of C. cauliflora fruit extract on developing chick embryos. Materials and Methods: Three hundred fertilized eggs were divided into 2 groups; 1) the experimental groups received various doses of C. cauliflora fruit extract at 50, 100, 200, and 400 mg/kg, and 2) the control group received a 0.9 % normal saline solution. One hundred and fifty eggs at embryonic day-3 (ED-3) were observed for LC50, morphology, and histology, while the 150 eggs at embryonic day-6 (ED-6) were investigated for morphology and histology. Results: At a dosage of 291.70 mg/kg, the C. cauliflora fruit extract induced mortality in ED-3 by more than 50 %. Moreover, fruit extraction can also result in anomalies in ED-3 and ED-6, such as anterior neuropore openings, heart tubes, liver and kidney tissue defects. Conclusion: As a result, it was determined that tissue damage and organ malformation followed dose-dependent and all-or-none laws. Despite having a wide range of medical properties, C. cauliflora fruit extracts have been demonstrated to impair early growth and development and cause birth abnormalities in animal studies. This will allow further determination of regulatory criteria for medical use, particularly for pregnant women. HIGHLIGHTS First report of the cauliflora fruit extract induced embryonic lethality The cauliflora fruit extract induces embryonic organ and tissue anomalies following dose-dependent and all-or-none laws These experimental protocols are beneficial for teratogenesis studies GRAPHICAL ABSTRACT

Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly.

As the microtubule-organizing centers of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and microtubule-organizing center function. Here, we investigate the post-transcriptional regulation of Plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the Plp 3'-untranslated region and examine the role of the CPEB ortholog Oo18 RNA-binding protein (Orb) in Plp mRNA regulation. Our data show that Orb interacts biochemically with Plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and its function in PCM scaffolding are compromised in orb mutant embryos, resulting in genomic instability and embryonic lethality. Moreover, we find that PLP overexpression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. Our data suggest that Orb modulates PLP expression at the level of Plp mRNA polyadenylation and demonstrates that the post-transcriptional regulation of core, conserved centrosomal mRNAs is crucial for centrosome function.

PQN-59 antagonizes microRNA-mediated repression during post-embryonic temporal patterning and modulates translation and stress granule formation in C. elegans.

microRNAs (miRNAs) are potent regulators of gene expression that function in a variety of developmental and physiological processes by dampening the expression of their target genes at a post-transcriptional level. In many gene regulatory networks (GRNs), miRNAs function in a switch-like manner whereby their expression and activity elicit a transition from one stable pattern of gene expression to a distinct, equally stable pattern required to define a nascent cell fate. While the importance of miRNAs that function in this capacity are clear, we have less of an understanding of the cellular factors and mechanisms that ensure the robustness of this form of regulatory bistability. In a screen to identify suppressors of temporal patterning phenotypes that result from ineffective miRNA-mediated target repression, we identified pqn-59, an ortholog of human UBAP2L, as a novel factor that antagonizes the activities of multiple heterochronic miRNAs. Specifically, we find that depletion of pqn-59 can restore normal development in animals with reduced lin-4 and let-7-family miRNA activity. Importantly, inactivation of pqn-59 is not sufficient to bypass the requirement of these regulatory RNAs within the heterochronic GRN. The pqn-59 gene encodes an abundant, cytoplasmically-localized, unstructured protein that harbors three essential “prion-like” domains. These domains exhibit LLPS properties in vitro and normally function to limit PQN-59 diffusion in the cytoplasm in vivo. Like human UBAP2L, PQN-59’s localization becomes highly dynamic during stress conditions where it re-distributes to cytoplasmic stress granules and is important for their formation. Proteomic analysis of PQN-59 complexes from embryonic extracts indicates that PQN-59 and human UBAP2L interact with orthologous cellular components involved in RNA metabolism and promoting protein translation and that PQN-59 additionally interacts with proteins involved in transcription and intracellular transport. Finally, we demonstrate that pqn-59 depletion reduces protein translation and also results in the stabilization of several mature miRNAs (including those involved in temporal patterning). These data suggest that PQN-59 may ensure the bistability of some GRNs that require miRNA functions by promoting miRNA turnover and, like UBAP2L, enhancing protein translation.

Studying the immune response genes expression regulation of Drosophila melanogaster

The main control of the synthesis of the antimicrobial peptides in the cells of Drosophila melanogaster is carried out by two signalling pathways IMD and Toll, which include the classic transcription factors of the NF-kB family (Dif, Dorsal and Relish). The Toll pathway is activated in response to gram-positive bacteria and fungi, and the IMD pathway is activated in response to gram-negative bacteria. In addition to transcription factors, the transcriptional apparatus of the cells includes the co-activators of transcription, such as the SAYP protein, which was described earlier in our laboratory. Co-activators do not bind directly to DNA, but assemble different parts of the transcriptional complex, which includes chromatin-remodelling complex, the specific transcription factors and the general transcription factors, which bind to RNA Pol II. SAYP homologues were found in the genomes of all multicellular animals. The protein mediates various functions of ontogenesis. We found that knockdown of SAYP leads to a decrease in expression of the specific genes of the IMD and Toll immune response pathways. In addition, the proteomic analysis of proteins associated with SAYP revealed sequences of another transcription factor, which is called DEAF-1. This protein is also involved in the immune response and ontogenesis. These prerequisites determined the task for studying the interactions of SAYP, Dif, Dorsal, Relish and DEAF-1 factors and their influence on the genes expression activation. We have obtained antibodies to the described factors. The most optimal epitopes (presumably exposed and accessible for recognition) were selected; the corresponding fragments of the cDNA genes were inserted into the expression vector and the conditions of expression in E.coli cells were matched. Recombinant proteins were produced and purified by metal-chelate affinity chromatography using Ni-Sepharose. The presence of these proteins in the fractions was confirmed by MALDI. The proteins have been used to immunize rabbits. The antibodies have been affinity purified from sera. The specificity of the antibodies was verified by Western blot analysis of the Drosophila embryonic nuclear extract. We have developed a model system for activating the IMD and Toll pathways. Escherichia coli and Micrococcus luteus cells were grown, washed and resuspended in water. The suspensions were inactivated and added to the S2 D. melanogaster cell cultures. Using qPCR, we have observed the activation of the immune response genes. Using the model system, we checked the recruitment of two model genes (Cecropin A1, Metchnikowin) to the promoters of the studied transcription co-activator. Chromatin immunoprecipitation has shown the binding of studied co-factor to gene promoters upon their activation. Immunoprecipitation using the obtained antibodies to DEAF-1 and SAYP has shown the co-precipitation of DEAF-1 protein with SAYP. This indicates that these proteins may jointly participate in the regulation of the immune response genes expression. We are currently looking for the individual domains of these proteins that mediate their interactions.

Embryonic stem cell extracts improve wound healing in diabetic mice.

Impaired wound healing significantly impacts morbidity and mortality in diabetic patients, necessitating the development of novel treatments to improve the wound healing process. We here investigated the topical use of acellular embryonic stem cell extracts (EXTs) in wound healing in diabetic db/db mice. Wounds were induced in diabetic db/db mice, which were subsequently treated with EXTs, with 3T3 fibroblast cell line protein extracts (3T3XTs) or with saline as a control. Pathology and mechanistic assays were then performed. The in vivo topical administration of EXTs facilitates wound closure, contraction and re-epithelialization. Moreover, EXTs reduced the number of wound-infiltrating CD45+ inflammatory cells and increased the rate of repair and of angiogenesis as compared to controls. Interestingly, the EXT effect was partly enhanced by the use of a collagen-based biocompatible scaffold. In vivo, topical administration of EXTs increased the percentage of regulatory T cells in the wounded tissue, while in vitro EXT treatment reduced T cell-mediated IFN-γ production. Proteomic screening revealed 82 proteins differentially segregating in EXTs as compared to 3T3 extracts, with APEX1 identified as a key player for the observed immunomodulatory effect of EXTs. EXTs are endowed with immunoregulatory and anti-inflammatory properties; their use improves wound healing in diabetic preclinical models.

Specific PCR method for detection of species origin in biochemical drugs via primers for the ATPase 8 gene by electrophoresis

A PCR method is described to identify the species origin of various animal and human tissue-derived biochemical drugs. Four commercialized drugs, including spermary tablets, compound embryonic bovine liver extract tablets, spleen aminopeptide solution, and placenta polypeptide injection, were used as a proof-of-principle in this study. Primers were designed to amplify conservative regions of mitochondrial cytochrome b and ATPase 8 genes from beef, pork, lamb and human DNA, respectively. The specificity of primers for ATPase 8 gene is found to be higher than those for cytochrome b under the given experimental conditions. The amplicon sizes of ATPase 8 were 212, 271, 293 and 405bp for pork, beef, lamb and human tissue, respectively. The minimum detectable concentration of DNA sample for species identification is 0.05-0.5pg·μL-1. The species origin can be distinguished by this method in extremely low concentrations of template DNAs extracted. Conceivably, this PCR method for meat authentication may be extended to quality control of other biochemical drugs and raw materials. Graphical abstract A specific PCR method was developedfor thedetection of species origin in biochemical drugs via species-specific primers targeting mitochondrial ATPase 8 genes. The PCR products were separated by gel electrophoresis and species origins were indicated by comparison to references.

Early lysosomal maturation deficits in microglia triggers enhanced lysosomal activity in other brain cells of progranulin knockout mice

BackgroundHeterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear.MethodsmRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins.ResultsWe demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN.ConclusionsThe present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells.

Embryonic germ cell extracts erase imprinted genes and improve the efficiency of induced pluripotent stem cells

Patient-specific induced pluripotent stem cells (iPSCs) have the potential to be useful in the treatment of human diseases. While prior studies have reported multiple methods to generate iPSCs, DNA methylation continues to limit the totipotency and reprogramming efficiency of iPSCs. Here, we first show the competency of embryonic germ cells (EGCs) as a reprogramming catalyst capable of effectively promoting reprogramming induced by four defined factors, including Oct4, Sox2, Klf4 and c-Myc. Combining EGC extracts with these four factors resulted in formation of more embryonic stem cell-like colonies than did factors alone. Notably, expression of imprinted genes was higher with combined induction than with factors alone. Moreover, iPSCs derived from the combined inductors tended to have more global hypomethylation. Our research not only provides evidence that EGC extracts could activate DNA demethylation and reprogram imprinted genes, but also establishes a new way to enhance reprogramming of iPSCs, which remains a critical safety concern for potential use of iPSCs in regenerative medicine.

Embryonic Cell Extracts Ameliorate Wound Healing in Diabetic Mice

Wound healing (WH) is a physiological function in which a balanced inflammatory response is required for tissue regeneration and successful wound closure. In diabetic patients WH is impaired and negatively impacts diabetic individuals in terms of morbidity, mortality and social disability, while the lack of an effective wound care highlights the need for novel therapeutic strategies. Extracts obtained from embryonic stem cells (EXTs) exhibited immunomodulatory properties and we hypothesize that EXTs may improve WH in diabetes. Here we explored the in vitro and in vivo effect of EXTs on WH using the diabetic db/db mouse model. Our data show that in vivo EXT topical administration ameliorates wound closure, contraction and re-epithelization by significantly reducing local inflammation and enhancing angiogenesis as compared to acellular protein extracts from 3T3 fibroblast cells (3T3xt) or no treatment. EXTs increased the percentage of regulatory T cells in the wound in vivo and inhibited T cell activation and IFNγ production in vitro. Db/db-derived dendritic cells exposed to EXTs showed decreased maturation, with downregulation of the costimulatory molecules CD80, CD86 and CD40. On the other hand, in vivo 3T3xt administration caused poorer wound closure with enhanced leucocyte infiltration of wounds and CD80, CD86 and CD40 upregulation on activated dendritic cells both in vivo and in vitro. Finally, proteomic analysis revealed 82 molecular entities differentially segregating in the EXT and 3T3xt group. Following unbiased selection, 10 proteins were filtered and assayed for their immune activity. Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) revealed strong immunomodulatory properties at ELISpot analysis by inhibiting IFNγ secretion of human PBMCs. APEX1 may result as the key factor mediating the beneficial effect of EXT on WH. EXTs or acellular extracts with upregulated APEX1 is a tool of remarkable translational potential for a safe, innovative and cost-effective application in the clinic. Disclosure C. Loretelli: None. M. Ben Nasr: None. G. Al Haj: None. F. Rocchio: None. S. Faravelli: None. A. Malvandi: None. F. D'Addio: None. A. Maestroni: None. T. Letizia: None. E. Assi: None. V. Usuelli: None. E. Castillo Leon: None. R. Meroni: None. G. Zuccotti: None. P. Fiorina: None.

Inducing hair follicle neogenesis with secreted proteins enriched in embryonic skin

Organ development is a sophisticated process of self-organization. However, despite growing understanding of the developmental mechanisms, little is known about how to reactivate them postnatally for regeneration. We found that treatment of adult non-hair fibroblasts with cell-free extract from embryonic skin conferred upon them the competency to regenerate hair follicles. Proteomics analysis identified three secreted proteins enriched in the embryonic skin, apolipoprotein-A1, galectin-1 and lumican that together were essential and sufficient to induce new hair follicles. These 3 proteins show a stage-specific co-enrichment in the perifolliculogenetic embryonic dermis. Mechanistically, exposure to embryonic skin extract or to the combination of the 3 proteins altered the gene expression to an inductive hair follicle dermal papilla fibroblast-like profile and activated Igf and Wnt signaling, which are crucial for the regeneration process. Therefore, a cocktail of organ-specific extracellular proteins from the embryonic environment can render adult cells competent to re-engage in developmental interactions for organ neogenesis. Identification of factors that recreate the extracellular context of respective developing tissues can become an important strategy to promote regeneration in adult organs.

Eml1 loss impairs apical progenitor spindle length and soma shape in the developing cerebral cortex

The ventricular zone (VZ) of the developing cerebral cortex is a pseudostratified epithelium that contains progenitors undergoing precisely regulated divisions at its most apical side, the ventricular lining (VL). Mitotic perturbations can contribute to pathological mechanisms leading to cortical malformations. The HeCo mutant mouse exhibits subcortical band heterotopia (SBH), likely to be initiated by progenitor delamination from the VZ early during corticogenesis. The causes for this are however, currently unknown. Eml1, a microtubule (MT)-associated protein of the EMAP family, is impaired in these mice. We first show that MT dynamics are perturbed in mutant progenitor cells in vitro. These may influence interphase and mitotic MT mechanisms and indeed, centrosome and primary cilia were altered and spindles were found to be abnormally long in HeCo progenitors. Consistently, MT and spindle length regulators were identified in EML1 pulldowns from embryonic brain extracts. Finally, we found that mitotic cell shape is also abnormal in the mutant VZ. These previously unidentified VZ characteristics suggest altered cell constraints which may contribute to cell delamination.

Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition.

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations invitro and functional analysis of target genes invivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential invitro and invivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.

Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.

The diaphanous homologue Diaph3 (aka mDia2) is a major regulator of actin cytoskeleton. Loss of Diaph3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of actin in the cleavage furrow. Here we report that Diaph3 is required before cell fission, to ensure the accurate segregation of chromosomes. Inactivation of the Diaph3 gene causes a massive loss of cortical progenitor cells, with subsequent depletion of intermediate progenitors and neurons, and results in microcephaly. In embryonic brain extracts, Diaph3 co-immunoprecipitates with BubR1, a key regulator of the spindle assembly checkpoint (SAC). Diaph3-deficient cortical progenitors have decreased levels of BubR1 and fail to properly activate the SAC. Hence, they bypass mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation, generating aneuploidy. Our data identify Diaph3 as a major guard of cortical progenitors, unravel novel functions of Diaphanous formins and add insights into the pathobiology of microcephaly.

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila.

The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

Abstract 841: SNP-FEMS: a method to identify DNA binding proteins interacting with enhancer elements

Abstract An important goal of human genetics is to understand the etiology of genetic traits. In the past few years, genome-wide association studies (GWAS) have identified thousands of chromosomal loci associated with hundreds of traits. GWAS provide a list of variants (single nucleotide polymorphisms, SNP) that are statistically associated with a phenotype, but do not offer any direct evidence about the biological mechanism. Most GWAS cancer susceptibility loci are located in non-coding regions suggesting that they function by modifying tracsriptional regulatory elements, such as enhancers and promoters of target genes. Thus, methods to help identify transcription factors that interact with regulatory regions containing risk associated variants can reveal biological mechanisms. We developed a method, SNP-FEMS (FPLC, EMSA, Mass Spec) that combines liquid chromatography (FPLC), electrophoretic mobility shift assays (EMSAs), and mass spectrometry to identify transcription factors interacting with putative regulatory regions at the 8q24 ovarian cancer risk locus. Human embryonic kidney 293FT nuclear extract was subjected to Sepharose 6 10/300 GL size fractionation and all the fractions were screened for allele-specific binding proteins by EMSA using a probe containing a genome-wide significant SNP. Fractions with positive signal were combined, concentrated and further fractionated with an ion exchange column MonoQ 5/10 GL to further reduce the complexity of the fractions. Fractions were screened by EMSA with the same probe, and the positive fractions were combined, concentrated, subjected to PAGE, in-gel trypsin digestion, and analyzed by Mass Spectrometry to identify proteins in the fractions. Bioinformatics analysis predicted different transcription factors binding to the putative regulatory region and the only transcription factor that was also present in our experimental dataset was the Cyclin AMP-dependent transcription factor ATF-1. This data suggests that ATF1 is implicated in ovarian cancer susceptibility and demonstrate the utility of the SNP-FEMS method to identify regulatory factors involved in ovarian cancer risk. This technique is applicable to any predisposition loci mediated by changes in regulatory elements. Citation Format: Gustavo A. Mendoza-Fandino, Nicholas Woods, Rebekah Baskin, Anxhela Gjyshi, Alvaro N. Monteiro. SNP-FEMS: a method to identify DNA binding proteins interacting with enhancer elements. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 841. doi:10.1158/1538-7445.AM2015-841

Giving Protamines the Boot

Protamines wind sperm DNA up tight, making it sixfold more compact than a metaphase chromosome. Upon fertilization, these protamines must be jettisoned and replaced with histones—but exactly how has been unclear. A study in Drosophila melanogaster now identifies four factors present in the zygote that mediate this process. Previous studies have suggested that the removal of protamines and the assembly of histone-containing nucleosomes are distinct processes. For instance, two Drosophila factors have been identified that are required for nucleosome assembly after fertilization, but they are not involved in protamine removal. In this new study, Alexander Emelyanov et al. [1] developed an in vitro assay for Drosophila sperm chromatin remodeling—protamine removal and histone addition. Using a crude Drosphila embryonic extract, the researchers identified factors that remained bound to protamines removed from sperm chromatin, and zeroed in on four. They showed that these four proteins were sufficient in their assay to dissociate protamines from DNA. The researchers found that three of the proteins, NAP-1, NLP, and nucleophosmin, were involved in removing protamine A, one of two protamines in Drosophila melanogaster. TAP/p32, the fourth factor, removes protamine B; consistent with this role, the researchers found that embryos from P32-null females failed to properly form the male pronucleus. Moreover, TAP/p32 was also shown to have an additional role as a histone chaperone, mediating nucleosome assembly—a role previously also pinned to NAP-1, NLP, and nucleophosmin. The researchers speculate that protamine chaperones, such as these four, evolved from proteins with core functions in nucleosome assembly and remodeling. Consistent with that idea, they showed that the ortholog of TAP/p32 in C. cerevisiae—a species without protamines—is involved in chromatin remodeling and DNA compaction and/or repair. Protamines are highly divergent evolutionarily, and other species may use distinct protamine chaperones to accomplish the task of protamine removal.

Cis-regulatory control of the nuclear receptor Coup-TF gene in the sea urchin Paracentrotus lividus embryo.

Coup-TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well-studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup-TF gene (PlCoup-TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup-TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratus upstream Coup-TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis-acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site-specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup-TF gene at pluteus stage sea urchin embryos. These findings lead to the future identification of these factors and to the hierarchical positioning of PlCoup-TF within the embryonic GRN.

Vangl2 regulates E-cadherin in epithelial cells.

E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions.