Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced micro- RNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these prodifferentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.Comment: 45 pages 5 figure

Community Building im Bereich ePublishing

Im Zentrum dieses Vortrages steht das von der Deutschen Forschungsgemeinschaft geförderte CARPET Project (Community for Academic Reviewing, Publishing and Editorial Technology) (1), welches eine elektronische Plattform aufbaut, mit dem Ziel, einen zentralen Einstieg zum Thema ePublishing Software zu bieten. Im Fokus des CARPET Portals stehen ein Katalog mit der systematischen Darstellung von ePublishing Softwaretools und Services, sowie ein angehängtes Nutzerforum als Kollaborationsmittel. Um das Ziel von CARPET effektiver zu verfolgen, wird eine ePublishing Community aufgebaut, welche zunächst hauptsächlich aus den drei evaluierten Interessengruppen besteht: Entwickler, Anbieter und Nutzer von Softwaretools und Services. Diese Gruppen sollen über die CARPET Plattform im Diskurs zusammengebracht werden, um mit Ihrer Hilfe a) Redundanzen und Defiziten bei den bestehenden Entwicklungen besser zu identifizieren, b) die Nachhaltigkeit dieser Lösungen durch Nachnutzung und Weiterentwicklung zu sichern, c) ein möglichst breites Spektrum an ePublishing Nutzungsszenarien und Endnutzer-Anforderungen zu erkennen, sodass die Entwickler- und Anbietergruppen darauf reagieren können und d) Leitbilder und Standards zu identifizieren. In diesem Beitrag soll der oben beschriebene Vorgang, und insbesondere Methoden, Herausforderungen und erste Ergebnisse dargestellt und diskutiert werden. (1) Das CARPET Project ist eine Initiative der Humboldt Universität Berlin, der Staats- und Universitätsbibliothek Göttingen und der Max Planck Digital Library. Weitere Informationen unter: www.carpet-project.net

Normalising jurisdictional heterotopias through place branding : the cases of Christiania and Metelkova

This paper explores the political dimensions of place branding as a path to normalisation for areas where a paradoxical relationship with the law exists, places that we coin “jurisdictional heterotopias” borrowing from Foucauldian literature. We posit that place branding plays a fundamental role in facilitating scale jumping in the otherwise vertically aligned legal space, a hierarchy designed to exclude spatial multiplicity from its premise. By examining the role of place branding in such areas, we endeavour to understand and appreciate the selective application of the law, the perpetuation of unregulated and illegal activity, as well as the place – specificity of legal practice. Ultimately, we argue that strong place branding associations permit the engulfment of this type of heterotopias in the “mainstream” leading to their normalisation; such a normalisation results not only in the acceptance of their uniqueness by the institutional elements, but also in the potential nullification of the liberties their communities advocate

Achromobacter xylosoxidans respiratory tract infection in cystic fibrosis patients

The aims of this study were to evaluate the frequency of Achromobacter xylosoxidans infection in a cohort of cystic fibrosis patients, to investigate antimicrobial sensitivity, to establish possible clonal likeness among strains, and to address the clinical impact of this infection or colonization on the general outcome of these patients. The study was undertaken between January 2004 and December 2008 on 300 patients receiving care at the Regional Cystic Fibrosis Center of the Naples University “Federico II”. Sputum samples were checked for bacterial identification. For DNA fingerprinting, pulsed-field gel electrophoresis (PFGE) was carried out. Fifty-three patients (17.6%) had at least one positive culture for A. xylosoxidans; of these, 6/53 (11.3%) patients were defined as chronically infected and all were co-colonized by Pseudomonas aeruginosa. Of the patients, 18.8% persistently carried multidrug-resistant isolates. Macrorestriction analysis showed the presence of seven major clusters. DNA fingerprinting also showed a genetic relationship among strains isolated from the same patients at different times. The results of DNA fingerprinting indicate evidence of bacterial clonal likeness among the enrolled infected patients. We found no significant differences in the forced expiratory volume in 1 s (FEV1) and body mass index (BMI) when comparing the case group of A. xylosoxidans chronically infected patients with the control group of P. aeruginosa chronically infected patients

Dicer Is Associated with Ribosomal DNA Chromatin in Mammalian Cells

Background: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. Methodology/Principal Findings: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer 2/2 ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. Conclusion/Significance: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since th

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

MicroRNA profiling of rhesus macaque embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) play important roles in embryonic stem cell (ESC) self-renewal and pluripotency. Numerous studies have revealed human and mouse ESC miRNA profiles. As a model for human-related study, the rhesus macaque is ideal for delineating the regulatory mechanisms of miRNAs in ESCs. However, studies on rhesus macaque (r)ESCs are lacking due to limited rESC availability and a need for systematic analyses of fundamental rESC characteristics.</p> <p>Results</p> <p>We established three rESC lines and profiled microRNA using Solexa sequencing resulting in 304 known and 66 novel miRNAs. MiRNA profiles were highly conserved between rESC lines and predicted target genes were significantly enriched in differentiation pathways. Further analysis of the miRNA-target network indicated that gene expression regulated by miRNAs was negatively correlated to their evolutionary rate in rESCs. Moreover, a cross-species comparison revealed an overall conservation of miRNA expression patterns between human, mouse and rhesus macaque ESCs. However, we identified three miRNA clusters (miR-467, the miRNA cluster in the imprinted Dlk1-Dio3 region and C19MC) that showed clear interspecies differences.</p> <p>Conclusions</p> <p>rESCs share a unique miRNA set that may play critical roles in self-renewal and pluripotency. MiRNA expression patterns are generally conserved between species. However, species and/or lineage specific miRNA regulation changed during evolution.</p

miR-125b Promotes Early Germ Layer Specification through Lin28/let-7d and Preferential Differentiation of Mesoderm in Human Embryonic Stem Cells

Unlike other essential organs, the heart does not undergo tissue repair following injury. Human embryonic stem cells (hESCs) grow indefinitely in culture while maintaining the ability to differentiate into many tissues of the body. As such, they provide a unique opportunity to explore the mechanisms that control human tissue development, as well as treat diseases characterized by tissue loss, including heart failure. MicroRNAs are small, non-coding RNAs that are known to play critical roles in the regulation of gene expression. We profiled the expression of microRNAs during hESC differentiation into myocardial precursors and cardiomyocytes (CMs), and determined clusters of human microRNAs that are specifically regulated during this process. We determined that miR-125b overexpression results in upregulation of the early cardiac transcription factors, GATA4 and Nkx2-5, and accelerated progression of hESC-derived myocardial precursors to an embryonic CM phenotype. We used an in silico approach to identify Lin28 as a target of miR-125b, and validated this interaction using miR-125b knockdown. Anti-miR-125b inhibitor experiments also showed that miR-125b controls the expression of miRNA let-7d, likely through the negative regulatory effects of Lin28 on let-7. We then determined that miR-125b overexpression inhibits the expression of Nanog and Oct4 and promotes the onset of Brachyury expression, suggesting that miR-125b controls the early events of human CM differentiation by inhibiting hESC pluripotency and promoting mesodermal differentiation. These studies identified miR-125b as an important regulator of hESC differentiation in general, and the development of hESC-derived mesoderm and cardiac muscle in particular. Manipulation of miR-125b-mediated pathways may provide a novel approach to directing the differentiation of hESC-derived CMs for cell therapy applications