Stabilization of ß-Catenin Directs HEB to Limit Thymic Selection.

Activation of ß-catenin in CD4+CD8+ double-positive (DP) thymocytes halts development before the thymic selection stage and predisposes to transformation. Leukemogenesis, but not the developmental block, depends on TCF-1, ß-catenin's DNA-binding partner. In this study, we show that ß-catenin activation directs the DNA-binding protein HEB to block DP thymocyte development. Conditional loss of HEB in DP thymocytes with stabilized ß-catenin restores the frequencies of postselection TCRßhi/CCR7+ and TCRßhi/CD69+ DPs and their cell-cycle profile. This recovery is associated with significant reversal of ß-catenin-induced expression changes, particularly those related to the CD69+ DP cell signature and to cell-cycle pathways. Stabilizing ß-catenin in DP thymocytes directs HEB binding to ≈11,000 novel DNA sites throughout the genome. Novel HEB sites mark most CD69+ DP cell signature genes that change expression upon activation of ß-catenin and then revert after loss of HEB. Moreover, many of the novel HEB sites occupy promoter regions of genes enriched in mitotic cell cycle pathways. HEB binding to those regions correlates with downregulation of the associated genes, and HEB inactivation restores expression to physiologic levels. These findings highlight a molecular interplay between HEB and ß-catenin that can impair thymic development.

Effects of the Essential Oil from Pistacia lentiscus Var. chia on the Lateral Line System and the Gene Expression Profile of Zebrafish (Danio rerio).

Mastic essential oil exhibits anti-bacterial, anti-inflammatory, and anti-oxidant properties. With the growing interest of the use of mastic oil in the food and pharmaceutical industry, systematic in vivo studies are needed to address controlled usage and safety issues. In the present work we evaluated the safety of mastic oil using as a model the zebrafish lateral line system. In addition, we studied the gene expression profile of zebrafish fed with mastic oil-supplemented diet using microarray analysis. Our results showed that the hair cells of lateral line neuromasts are functional upon exposure of zebrafish larvae up to 20 ppm of mastic essential oil, while treatment with higher concentrations, 100 and 200 ppm, resulted in increased larvae mortality. Dietary supplementation of zebrafish with mastic essential oil led to differential expression of interferon response-related genes as well as the immune responsive gene 1 (irg1) that links cellular metabolism with immune defense. Notably, mucin 5.2, a constituent of the mucus hydrogel that protects the host against invading pathogens, was up-regulated. Our in vivo work provides information concerning the safety of mastic essential oil use and suggests dietary effects on gene expression related with the physical and immunochemical properties of the gastrointestinal system.

The zebrafish homologs of SET/I2PP2A oncoprotein: expression patterns and insights into their physiological roles during development.

The oncoprotein SET/I2PP2A (protein phosphatase 2A inhibitor 2) participates in various cellular mechanisms such as transcription, cell cycle regulation and cell migration. SET is also an inhibitor of the serine/threonine phosphatase PP2A, which is involved in the regulation of cell homeostasis. In zebrafish, there are two paralogous set genes that encode Seta (269 amino acids) and Setb (275 amino acids) proteins which share 94% identity. We show here that seta and setb are similarly expressed in the eye, the otic vesicle, the brain and the lateral line system, as indicated by in situ hybridization labeling. Whole-mount immunofluorescence analysis revealed the expression of Seta/b proteins in the eye retina, the olfactory pit and the lateral line neuromasts. Loss-of-function studies using antisense morpholino oligonucleotides targeting both seta and setb genes (MOab) resulted in increased apoptosis, reduced cell proliferation and morphological defects. The morphant phenotypes were partially rescued when MOab was co-injected with human SET mRNA. Knockdown of setb with a transcription-blocking morpholino oligonucleotide (MOb) resulted in phenotypic defects comparable with those induced by setb gRNA (guide RNA)/Cas9 [CRISPR (clustered regularly interspaced short palindromic repeats)-associated 9] injections. In vivo labeling of hair cells showed a significantly decreased number of neuromasts in MOab-, MOb- and gRNA/Cas9-injected embryos. Microarray analysis of MOab morphant transcriptome revealed differential expression in gene networks controlling transcription in the sensory organs, including the eye retina, the ear and the lateral line. Collectively, our results suggest that seta and setb are required during embryogenesis and play roles in the zebrafish sensory system development.

Transcriptional and Behavioral Responses of Zebrafish Larvae to Microcystin-LR Exposure.

Microcystins are cyclic heptapeptides that constitute a diverse group of toxins produced by cyanobacteria. One of the most toxic variants of this family is microcystin-LR (MCLR) which is a potent inhibitor of protein phosphatase 2A (PP2A) and induces cytoskeleton alterations. In this study, zebrafish larvae exposed to 500 µg/L of MCLR for four days exhibited a 40% reduction of PP2A activity compared to the controls, indicating early effects of the toxin. Gene expression profiling of the MCLR-exposed larvae using microarray analysis revealed that keratin 96 (krt96) was the most downregulated gene, consistent with the well-documented effects of MCLR on cytoskeleton structure. In addition, our analysis revealed upregulation in all genes encoding for the enzymes of the retinal visual cycle, including rpe65a (retinal pigment epithelium-specific protein 65a), which is critical for the larval vision. Quantitative real-time PCR (qPCR) analysis confirmed the microarray data, showing that rpe65a was significantly upregulated at 50 µg/L and 500 µg/L MCLR in a dose-dependent manner. Consistent with the microarray data, MCLR-treated larvae displayed behavioral alterations such as weakening response to the sudden darkness and hypoactivity in the dark. Our work reveals new molecular targets for MCLR and provides further insights into the molecular mechanisms of MCLR toxicity during early development.

The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function.

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP-mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP-His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP-mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence.

Knockdown of prothymosin α leads to apoptosis and developmental defects in zebrafish embryos.

Prothymosin alpha (ProTα) is an abundant nuclear protein involved in cellular processes intricately linked to development, such as cell proliferation and apoptosis. Although it is known that ProTα inhibits the formation of apoptosome and blocks caspase-3 activity, its mechanism of function in the apoptotic machinery is still under investigation. We have studied the cellular role of ProTα by knocking down its expression in HeLa cells with small hairpin RNA (shRNA) in the absence of apoptotic stimuli. Flow cytometric analysis showed that the live cell population was significantly decreased with a concomitant increase of the apoptotic populations. To understand the physiological role of ProTα within the context of embryonic development, we knocked down the Ptmab zebrafish ortholog using 2 specific morpholino oligonucleotides. Ptmab morphants exhibited growth retardation, bended trunks, and curly tails. The frequency of occurrence of the phenotypic defects was increased in a morpholino dose-dependent manner. Co-injection of ptmaa mRNA with ptmab morpholino partially rescued the morphological defects. Immunostaining with the anti-phospho-histone H3 (pH3) antibody suggested that the abnormalities of Ptmab morphants could be due to defective cell proliferation that results in growth imbalances. TUNEL fluorescent labelling and Acridine Orange staining of the morphants showed high rates of cell death in the head and tail regions. Concomitantly, the active form of caspase-3 was detected in Ptmab morphants. Our data suggest a conserved anti-apoptotic role of ProTα between zebrafish and humans, and provide the first evidence that ProTα is important for early embryogenesis.

Targeting of SET/I2PP2A oncoprotein inhibits Gli1 transcription revealing a new modulator of Hedgehog signaling.

The Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.

RNA association or phosphorylation of the RS domain prevents aggregation of RS domain-containing proteins.

Domains rich in alternating arginine and serine residues (RS domains) are found in a large number of eukaryotic proteins involved in several cellular processes. According to the prevailing view RS domains function as protein interaction domains, thereby promoting the assembly of higher-order cellular structures. Furthermore, recent data demonstrated that the RS regions of several SR splicing factors directly contact the pre-mRNA in a nonsequence specific but functionally important fashion. Using a variety of biochemical approaches, we now demonstrate that the RS domains of three proteins, not directly associated with the splicing reaction, such as lamin b receptor, acinus and peroxisome proliferator-activated receptor gamma coactivator-1 alpha, associate mainly with nuclear RNA and that this association is conducive in retaining the proteins in a soluble form. Phosphorylation by SRPK1 prevents RNA association, yet it greatly increases the fraction of the proteins recovered in soluble form, thereby mimicking the RNA effect. Based on these results we propose that the tendency to self-associate and form aggregates is a general property of RS domain-containing proteins and could be attributed to their disordered structure. RNA binding or SRPK1-mediated phosphorylation prevents aggregation and may serve to modulate the RS domain interaction modes.

Identification of distinct SET/TAF-Ibeta domains required for core histone binding and quantitative characterisation of the interaction.

BACKGROUND: The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Ibeta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Ibeta, we designed several SET/TAF-Ibeta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. RESULTS: Wild type SET/TAF-Ibeta binds to histones H2B and H3 with Kd values of 2.87 and 0.15 microM, respectively. The preferential binding of SET/TAF-Ibeta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Ibeta, as well as the H3 amino-terminal tail, are dispensable for this interaction. CONCLUSION: This type of analysis allowed us to assess the relative affinities of SET/TAF-Ibeta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Ibeta and can be valuable to understand the role of SET/TAF-Ibeta in chromatin function.

Prothymosin alpha associates with the oncoprotein SET and is involved in chromatin decondensation.

Prothymosin alpha (ProTalpha) is a histone H1-binding protein that interacts with the transcription coactivator CREB-binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two-hybrid assays, we show here that ProTalpha forms a complex with the oncoprotein SET. ProTalpha efficiently decondenses human sperm chromatin, while overexpression of GFP-ProTalpha in mammalian cells results in global chromatin decondensation. These results indicate that decondensation of compacted chromatin fibers is an important step in the mechanism of ProTalpha function.

The histone chaperone SET/TAF-Ibeta interacts functionally with the CREB-binding protein.

The oncoprotein SET/TAF-Ibeta is a histone chaperone which is involved in cell-cycle control and chromatin remodeling. Confocal laser scanning microscopy reveals that SET is localized in distinct foci of variable size throughout the nucleoplasm of interphase cells. We report here that SET interacts directly with the acetyltransferase CREB-binding protein (CBP) and enhances the transactivation potential of the transcription coactivator. Our data suggest that the histone chaperone SET regulates the CBP-mediated transcription and may indicate a general principle by which transcriptional regulators cooperate with histone chaperones for gene activation.

Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure.

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39.

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.

Prothymosin alpha interacts with the CREB-binding protein and potentiates transcription.

Prothymosin alpha (ProTalpha) is a histone H1-binding protein localized in sites of active transcription in the nucleus. We report here that ProTalpha physically interacts with the CREB-binding protein (CBP), which is a versatile transcription co-activator. Confocal laser scanning microscopy reveals that ProTalpha partially colocalizes with CBP in discrete subnuclear domains. Using transient transfections, we show that ProTalpha synergizes with CBP and stimulates AP1- and NF-kappaB-dependent transcription. Furthermore, overexpression of ProTalpha enhances the transactivation potential of CBP. These findings reveal a new function for ProTalpha in transcription activation, probably through CBP-mediated recruitment to different promoters.