CLIP and complementary methods

RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications

The SIB Swiss Institute of Bioinformatics' resources: focus on curated databases

The SIB Swiss Institute of Bioinformatics (www.isb-sib.ch) provides world-class bioinformatics databases, software tools, services and training to the international life science community in academia and industry. These solutions allow life scientists to turn the exponentially growing amount of data into knowledge. Here, we provide an overview of SIB's resources and competence areas, with a strong focus on curated databases and SIB's most popular and widely used resources. In particular, SIB's Bioinformatics resource portal ExPASy features over 150 resources, including UniProtKB/Swiss-Prot, ENZYME, PROSITE, neXtProt, STRING, UniCarbKB, SugarBindDB, SwissRegulon, EPD, arrayMap, Bgee, SWISS-MODEL Repository, OMA, OrthoDB and other databases, which are briefly described in this article

Persistent fetal γ-globin expression in adult transgenic mice following deletion of two silencer elements located 3′ to the human Aγ-globin gene

Natural deletions of the human γ-globin gene cluster lead to specific syndromes characterized by increased production of fetal hemoglobin in adult life and provide a useful model to delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. A hypothesis accounting for these phenotypic features assumes that silencers located within the Aγ-to δ-gene region are deleted in hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemias, leading to failure of switching. In the present study, we sought to clarify the in vivorole of two elements, termed Enh and F, located 3′ to the Aγ-globin, in silencing the fetal genes. To this end, we generated three transgenic lines using cosmid constructs containing the full length of the globin locus control region (LCR) linked to the 3.3-kb Aγ-gene lacking both the Enh and F elements. The Enh/F deletion resulted in high levels of Aγ-globin gene expression in adult mice in all single copy lines, whereas, the LCR-Aγ single copy lines which retain the Enh and F elements exhibited complete normal switching of the fetal Aγ-gene. Our study documents directly for the first time the in vivorole of these two gene-proximal negative regulatory elements in silencing the fetal globin gene in the perinatal period, and thus these data may permit their eventual exploitation in therapeutic approaches for thalassemias. © 2009 The Feinstein Institute for Medical Research

Identification of a STAT5 Target Gene, Dpf3, Provides Novel Insights in Chronic Lymphocytic Leukemia

STAT5 controls essential cellular functions and is encoded by two genes, Stat5a and Stat5b. To provide insight to the mechanisms linking hematologic malignancy to STAT5 activation/regulation of target genes, we identified STAT5 target genes and focused on Dpf3 gene, which encodes for an epigenetic factor. Dpf3 expression was induced upon IL-3 stimulation in Ba/F3 cells, while strong binding of both STAT5a and STAT5b was detected in its promoter. Reduced expression of Dpf3 was detected in Ba/F3 cells with Stat5a and Stat5b knock-down, suggesting that this gene is positively regulated by STAT5, upon IL-3 stimulation. Furthermore, this gene was significantly up-regulated in CLL patients, where DPF3 gene/protein up-regulation and strong STAT5 binding to the DPF3 promoter, correlated with increased STAT5 activation, mainly in non-malignant myeloid cells (granulocytes). Our findings provide insights in the STAT5 dependent transcriptional regulation of Dpf3, and demonstrate for the first time increased STAT5 activation in granulocytes of CLL patients. Novel routes of investigation are opened to facilitate the understanding of the role of STAT5 activation in the communication between non-malignant myeloid and malignant B-cells, and the functions of STAT5 target genes networks in CLL biology. © 2013 Theodorou et al

Pregnancy in patients with well-treated beta-thalassemia: Outcome for mothers and newborn infants

OBJECTIVE: Our purpose was to investigate the course and outcome of pregnancy in women with well-treated beta-thalassemia. STUDY DESIGN: Twenty-two pregnancies, including one twin pregnancy, in 19 women were studied. Pregnancy was advised when patients had received a prolonged intensive treatment with hypertransfusions and iron chelation and had echocardiographically normal resting left ventricular performance. All conceptions were spontaneous. Cardiac function, along with hematologic, endocrinologic, and hepatic parameters were initially assessed and monitored throughout pregnancy and for 2 to 9 years post: partum. Babies were delivered by elective cesarean section. RESULTS: Twenty-one healthy newborn infants were delivered. A spontaneous abortion and a case of exomphalos also occurred. Gestation, delivery, and recovery were surprisingly uneventful, and no significant cardiac complications were encountered. CONCLUSION: Pregnancy can be safe for mothers and babies, provided that women with thalassemia have been started early on intensive treatment and have a normal resting cardiac performance

Dynamic expression of the vertebrate-specific protein Nucks during rodent embryonic development

The nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS) is a highly phosphorylated nuclear protein that is overexpressed in many types of cancer. The flexibility of NUCKS and its extensive posttranslational modifications indicate that it is multifunctional, and its expression in most cell types suggests a housekeeping function. However, spatiotemporal expression of the Nucks protein during rodent development has not been reported. Thus, we investigated the expression of both the Nucks mRNA and protein during rat and mouse development by immunohistochemistry, in situ hybridization, Western immunoblotting, and reverse-transcription PCR analysis. We also used BLAST analysis against expressed sequence tag databases to determine whether a NUCKS homologue is expressed in invertebrate organisms. We found that Nucks expression increased during the initial stages of embryonic development, and then gradually decreased until birth in all tissues except the nervous tissue and muscle fibers. Interestingly, the expression of Nucks was very strong in migrating neural crest cells at E13.5 and ectoderm-derived tissues. In most tissues analyzed, the levels of Nucks correlated with the levels of Bax and activated caspase-3, which are indicative of apoptosis. Moreover, Nucks was upregulated very early during neuronal apoptosis in vitro. Expression analysis revealed that no transcript with close homology to the Nucks gene was present in invertebrates. The expression of Nucks in both proliferating and quiescent cells and its correlation with Bax levels and apoptosis strongly suggest that Nucks plays complex roles in cell homeostasis. Furthermore, the lack of homology in invertebrate organisms indicates a specific role for Nucks in vertebrate embryogenesis. © 2013 Elsevier Ltd. All rights reserved

Signs of striatal dopamine transporter density increase in association with improvement of tardive dyskinesia in a patient with schizophrenia, as demonstrated by a DAT scan

The imaging of the dopamine transporter could demonstrate the implication of dopaminergic pathway in the appearance of tardive dyskinesia. We report a case with psychotic and tardive dyskinesia symptoms. A DAT scan showed decreased dopamine transporter uptake in the area of brain's basal gaglia. A trial with quetiapine improved both psychotic and TD symptoms while a second DAT scan showed improvement status. We conclude that increased dopamine transporter uptake seemed to associate with the improvement of TD. © 2009 Elsevier B.V. All rights reserved

An embryonic-specific repressor element located 3 ` to the (A)gamma-globin gene influences transcription of the human beta-globin locus in transgenic mice

Objective. Persistent expression of the human fetal gamma-globin genes in the adult stage is often associated with naturally occurring deletions in the human beta-globin locus. The mapping of the 5’ breakpoints of these deletions within the (A)gamma- to delta-globin intergenic region has suggested that regulatory elements involved in the silencing of the gamma-globin genes in the adult may be present. We previously identified two elements in this region, termed Enh and F, located 3’ to the (A)gamma-globin gene acting as silencers in transient transfection assays. Here, we tested directly the in vivo significance of these elements in the developmental regulation of the human beta-like globin genes. Materials and Methods. We selectively deleted both Enh and F elements in the context of a 185-kb human beta-globin locus PAC (P1 phage artificial chromosome) and tested the effects of this deletion on the expression of the human beta-like globin genes in transgenic mice. Results. The Enh/F deletion resulted in an increase in epsilon- and gamma-globin mRNA levels in the embryonic yolk sac stage of erythropoiesis, which appears to be due to an increase in the rate of transcription rather than to an increase in the number of cells transcribing the human globin locus. However, the human developmental switching from fetal gamma-globin to adult beta-globin gene expression in transgenic mice was not affected by this deletion. Conclusion. These results identify Enh and F as locus-wide regulatory elements capable of down-regulating transcription of the human beta-globin locus in an embryonic-specific manner. (C) 2004 International Society for Experimental Hematology. Published by Elsevier Inc