Expression und Funktion des fettmasse- und adipositas-assoziierten Gens FTO

Expression und Funktion des fettmasse- und adipositas-assoziierten Gens FTO Genomweite Assoziationsstudien haben eine starke Assoziation zwischen einem Block von Einzelnukleotid Variationen (single nucleotide polymorphisms - SNPs) im Intron 1 des Fettmasse und Adipositas-assoziierten Gens (FTO), dem body mass index (BMI) und anderen, Adipositas bezogenen Erkrankungen bei Kindern und Erwachsenen vieler verschiedener Populationen gezeigt. Dennoch ist bisher nicht bekannt, wie stark der Effekt dieser Variationen auf die Expression von FTO und/oder anderen Genen ist. Darüber hinaus ist die biologische Funktion von FTO, insbesondere in Bezug auf die Regulation des Körpergewichts, noch immer Gegenstand intensiver Forschung. Das Ziel dieser Arbeit war die Untersuchung des Effekts des FTO Genotyps auf die Expression von FTO sowie die Aufklärung der Funktion des FTO Proteins durch Bestimmung der subzellularen Lokalisation und des Effekts der FTO Dosis auf RNA Expressionsprofile und RNA Modifizierungslevel. Daher wurden Expressionsstudien durchgeführt, um den Zusammenhang zwischen Adipositas-assoziierten SNPs und der Expression von FTO und/oder anderen Genen zu untersuchen sowie funktionelle Studien, um Einblick in die Biologie von FTO zu erlangen. Um die Frage zu klären, ob Adipositas-assoziierte Variationen die Transkription von FTO und/oder anderen Genen in cis beeinflussen, wurden Allel-spezifische Expressionsstudien mittels Primer-Extensions Assays genutzt. In verschiedenen Zelltypen konnte gezeigt werden, dass vom Risiko-Allel des FTO Gens ca. 40% mehr Transkript generiert wird als vom Nicht-Risiko-Allel. Die Charakterisierung einzelner SNPs im Hinblick auf ihre Lokalisation (in silico Ansatz) und Proteinbindeaktivität wies auf eine komplexe Regulation der FTO Expression hin. Dies wurde auch durch die Tatsache unterstützt, dass der zelluläre FTO mRNA Level durch eine Reihe von Transkriptionsfaktoren kontrolliert wird. Weiterhin konnte gezeigt werden, dass die Allel-spezifische Expression der benachbarten Gene RPGRIP1L und RBL2 unabhängig vom FTO Genotyp ist. Zur weiteren Klärung der Funktion des FTO Proteins, wurde der Effekt eines veränderten Protein Gehalts von FTO auf das Transkriptom und die RNA Methylierung untersucht. FTO Überexpression führte zu Veränderungen der steady-state Level von Genen, die bei der RNA Prozessierung und Metabolisierung eine Rolle spielen. Ein Mangel an FTO andererseits wirkte sich auf die Transkriptlevel von Genen aus, die bei der Zellantwort auf Nährstoffmangel beteiligt sind. Untersuchungen zur subzellularen Lokalisation zeigten, dass FTO vermehrt in nuclear speckles (punktförmigen Gebilden im Zellkern) vorkommt werden konnte, in denen RNA Spleißfaktoren gespeichert und modifiziert werden. Außerdem ist FTO in den Nucleoli vorhanden, wo ribosomale RNA transkribiert und prozessiert wird. In vitro Studien hatten Hinweise darauf geliefert, dass FTO als Nukleinsäure Demethylase agiert und dabei Einzelstrang RNA als Substrat bevorzugt. Daher wurden die Effekte von FTO auf RNA Methylierung untersucht. Durch den Vergleich des Gehalts von modifizierten und nicht-modifizierten Ribonukleosiden in RNA aus Gehirn von Fto-defizienten und Wildtyp Mäusen, konnte gezeigt werden, dass der FTO Gehalt das Verhältnis von 3-Methyluridin/Uridin and Pseudouridin/Uridin beeinflusst. In dieser Arbeit konnte ich zeigen, dass eine erhöhte Expression von FTO eine Prädisposition für Adipositas darstellt, möglicherweise durch Einfluss auf das Transkriptom und RNA Modifizierung. Weitere Untersuchungen werden dabei helfen, den Zusammenhang zwischen der Funktion von FTO, RNA Prozessierung und Adipositas weiter aufzuklären.Expression and Function of the Fat Mass and Obesity-Associated Gene FTO Genome-wide association studies have revealed a strong association between a block of single-nucleotide polymorphisms (SNPs) in intron 1 of the fat mass and obesity-associated (FTO) gene, body mass index (BMI) and other obesity-related traits in children and adults of many different populations. Yet, the impact of these variations on expression of FTO and/or other genes has remained unknown. Moreover, the biological function of FTO, in particular its contribution to body weight regulation, is still a subject of extensive investigations. The aim of this thesis was to investigate the impact of FTO genotype on FTO expression and elucidate the function of the FTO protein by determining its subcellular localization and the effect of FTO dosage on RNA expression profiles and RNA modification levels. Hence, expression studies were performed to evaluate the link between obesity-associated SNPs and expression of FTO and/or other genes, and functional studies were performed to gain insight into FTO biology. Allelic expression studies by primer extension assays were carried out to address the question whether obesity-associated variation affects transcription of the FTO and/or other genes in cis. It was demonstrated that the risk allele of FTO makes about 40% more transcripts than the non-risk allele in the different cell types. Characterization of single polymorphisms with regard to their location (in silico approach) and protein binding activity pointed to a complex regulation of the expression of FTO. This was strengthened by the fact that the cellular the level of FTO mRNA is controlled by a number of transcription factors. Allelic expression of the neighboring RPGRIP1L and RBL2 was shown to be independent of the FTO genotype. To elucidate the function of the FTO protein, effects of its altered levels on the transcriptome and RNA methylation were investigated. Overexpression of FTO resulted in changes of steady state levels of genes involved in RNA processing and metabolism, whereas deficiency of FTO led to alterations in transcripts levels of genes determining cellular response to starvation. Subcellular localization studies showed that FTO is enriched in nuclear speckles, where RNA splicing factors are stored and modified, and is present in nucleoli, where ribosomal RNA is transcribed and processed. In vitro studies have suggested that FTO acts as a nucleic acid demethylase and prefers single stranded RNA as a substrate. Therefore, the effects of FTO on RNA methylation were investigated. By comparison of content of modified and non-modified ribonucleosides in total brain RNA of Fto-deficient and wild type mice I could show that the level of FTO affects the 3-methyluridine/uridine and pseudouridine/uridine ratios. In summary, I could show that increased expression of FTO predisposes to obesity, possibly by affecting transcriptome and RNA modifications. Further investigations will help to elucidate the link between FTO function, RNA processing and obesity

Epigenetic dynamics of monocyte-to-macrophage differentiation

Background Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. Results Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. Conclusions In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation

The Human Retinoblastoma Gene Is Imprinted

Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. To date, ∼60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5′-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2′-deoxycytidine–treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation

N6-Adenosine Methylation in MiRNAs

<div><p>Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression.</p></div

The Coding and Small Non-coding Hippocampal Synaptic RNAome

Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more recently in the pre- and postsynaptic compartments as well. Other RNA species such as microRNAs have also been described at synapses where they are believed to control mRNA availability for local translation. A combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is, however, still lacking. Here, we isolate synaptosomes from the hippocampus of young wild-type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach for analyzing the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome, and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, our findings also support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable for studying the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and point to several important targets for further research

<i>K</i>nockdown of <i>FTO</i> does not significantly change mRNA levels of genes involved in miRNA biogenesis.

<p>The steady-state mRNA levels of <i>DICER</i>, <i>DROSHA</i>, <i>DGCR8</i> and <i>ADAR</i> were analyzed by qRT-PCR in cells treated with scrambled (scr) and <i>FTO-</i>specific siRNAs, respectively. <i>GAPDH</i> was used as a reference gene. The observed changes were not significant. Merged values of mean ± SD from triplicates per assay for the three independent cell lines FTO1C1, FTO2D4 and FTO3C3 are depicted. <i>FTO</i> kd, <i>FTO</i>-specific siRNA treated cells, scr siRNA, scrambled siRNA treated cells.</p

Deregulation of miRNAs in <i>FTO</i> knockdown cells.

<p>Mature miRNAs showing increased (A) and decreased (B) steady state levels in <i>FTO</i> knockdown cells. Normalized RNA-seq read numbers of individual miRNAs in <i>FTO</i> knockdown and scrambled siRNA treated cells were compared. Mean ± SD of three independent experiments are depicted. For verification and further studies, qRT-PCR analyses of selected mature miRNAs (C) and primary miRNA transcripts (D) were performed. We did not use other small RNAs as a reference gene for measuring mature miRNAs levels (as suggested by Life Technologies), since depletion of <i>FTO</i> might have an impact on their levels. Therefore, luciferase RNA was used to generate a standard curve and added to the qRT-PCR assays. <i>GAPDH</i> was used as a reference gene for measuring primary miRNAs transcript levels. Mean ± SD from quadruplicates per assay for three independent cell lines (FTO1C1, FTO2D4 and FTO3C3) are depicted. kd, <i>FTO</i> specific siRNA treated cells, scr, scrambled siRNA treated cells.</p

Effect of <i>FTO</i> knockdown on the steady state levels of methylated miRNAs.

<p>X-axis, log2 fold-changes (log2fc) of enrichment after imuunopreciptation with an anti-m6A antibody; y-axis, log2 fold-changes of steady state miRNA levels after <i>FTO</i> knockdown. The values of all 239 methylated miRNAs are shown. The red dotted line is the regression line.</p

miRNAs immunoprecipitated by the anti-m6A antibody.

<p>Seventeen miRNAs with >100 fold enrichment after immunoprecipitation in m6A RNA samples compared to IgG RNA samples are listed. Means and standard deviations of fold enrichments in three independent experiments are given. The complete list of immunoprecipated miRNAs can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118438#pone.0118438.s003" target="_blank">S3 Table</a>.</p><p>miRNAs immunoprecipitated by the anti-m6A antibody.</p