TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells

Comparative analysis of human and mouse transcriptomes of Th17 cell priming

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models

Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images

<p>Abstract</p> <p>Background</p> <p>Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed.</p> <p>Results</p> <p>To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies.</p> <p>Conclusions</p> <p>These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.</p

Transkription, kromatiinin ja soluviestinnän vuorovaikutusten analysointi tilastollisilla menetelmillä

Transcription is the first step in gene expression in which genetic information is transferred from DNA to RNA. Gene expression is highly controlled through transcriptional regulation at many steps. Transcriptional regulation in eukaryotes occurs, e.g., through binding of transcription factors and chromatin remodeling via various epigenetic pathways. Additionally, dysregulated transcription has been reported in various diseases. Thus, transcription and transcriptional regulation are of great interest for research. In this work, we study the transcriptome and its regulation using bioinformatic and computational biology approaches. We propose computational methods, LIGAP and DyNB, for analysis of temporal gene expression profiles measured using microarrays and RNA-seq, respectively. LIGAP is a methodology based on Gaussian processes for simultaneous differential expression analysis between an arbitratory number of time series microarray data sets. DyNB, is an extension of the Gaussian-Cox process in which the Poisson distribution is replaced by the negative binomial distribution. Additionally, DyNB enables the study of systematic differences, such as differential differentiation efficiencies, between conditions. Sorad, is a modeling framework based on differential equations and Gaussian processes for analysis of intracellular signaling transduction through phosphoprotein activities. We also propose and demonstrate how the in silico models inferred using Sorad can be used in estimating modulation strategies to obtain desired signaling response. Finally, we study the determinants of nucleosome positioning and subsequent effects on gene expression. All the proposed methods are benchmarked against existing methods and, in addition, they are applied to real-life problems. The comparison studies validate the applicability of the presented methods and demonstrate their improved performance relative to existing methods. Our transcriptome studies led to increased knowledge on the early differentiation of human T cells, and provided a valuable resource of candidate genes for future functional studies of the differentiation process. Our nucleosome study revealed that within loci important for T cell differentiation only 6% of the nucleosomes are differentially remodelled between T helper 1 and 2 cells and cytotoxic T lymphocytes. The remodelled nucleosomes correlated with the known differentiation program, chromatin accessibility, transcription factor binding, and gene expression. Finally, our data supports the hypothesis that transcription factors and nucleosomes compete for DNA occupancy.Geenin transkriptiossa kopioidaan DNA:ssa olevaa geneettistä koodia, joka johtaa geenien ilmentymiseen. Geenien ilmentymiseen johtava transkriptioaskel on tarkasti säädelty biologinen tapahtuma. Transkriptiota eukaryoottisoluissa säädellään muun muassa transkriptiotekijöiden sitoumisen promoottori- ja tehostaja-alueille ja epigeneettisten tekijöiden kautta. Geenien transkription säätelyn parempi ymmärtäminen on tärkeää, koska esimerkiksi transkription virheellinen säätely voi johtaa erilaisiin sairauksiin. Tämän väitöskirjan artikkeleissa on kehitetty laskennallisia menetelmiä geenien ilmentymisen ja ilmentymisen säätelyn tarkempaan tutkimiseen. LIGAP ja DyNB ovat gaussisiin prosesseihin perustuvia menetelmiä mikrosiruilla tai RNA-sekvenssoinnilla mitattujen aikasarja-aineistojen analysointiin. LIGAP-menetelmä soveltuu geenien ilmentymiserojen havainnointiin mielivaltaisessa määrässä biologisia näytteitä. DyNB-menetelmän tilastollinen malli voidaan nähdä Gaussin-Coxin prosessin laajennukseksi jossa Poissonin jakauma korvataan negatiivisella binomijakaumalla. DyNB-menetelmällä on mahdollista estimoida systemaattisia eroja näytteiden välillä. Solunsisäisten signaalinvälitysten tarkasteluun ja mallinnukseen kehitimme Sorad-menetelmän, joka perustuu differentiaaliyhtälöiden ja gaussisten prosessien yhdistämiseen. Sorad-menetelmä mahdollistaa myös analyysin, jossa estimoidaan miten ennalta määrättyjen komponenttien tulisi käyttäytyä jotta saadaan haluttu vaste aikaan. Väitöskirjan viimeisessä artikkelissa paikannamme nukleosomit tarkasti tutkiaksemme kromatiinin tilan vaikutusta geenien ilmentymiseen. Tekemämme vertailut aiempiin menetelmiin osoittivat kehitettyjen menetelmien edut. Tämän lisäksi kehitettyjä menetelmiä sovellettiin käytännön biologisiin ongelmiin. Geenien ilmentymisiä tarkastelleissa tutkimuksissa keskityimme napaverestä eristettyjen T-auttajasoluihin. Tuloksemme geenien ilmentymisestä T-auttajasolujen varhaisissa erilaistumisissa tarjoavat hyvän lähtökohdan tarkemmille jatkotutkimuksille. Nukleosomitutkimuksessamme osoitimme, että sytotoksisten T-solujen ja tyypin 1 ja T-auttajasolujen välillä ainoastaan kuudessa prosentissa nukleosomeista nähdään eroja. Havaitut muutokset nukleosomeissa korreloivat erilaistumisohjelman, avoimen kromatiinin, transkriptiotekijöiden sitoutumisen, ja geenien ilmentymisen kanssa. Lisäksi havaintomme tukevat hypoteesia nukleosomien ja transkriptiotekijöiden välisestä kilpailusta DNA:han sitoumisessa

LuxGLM

Motivation: 5-methylcytosine (5mC) is a widely studied epigenetic modification of DNA. The ten-eleven translocation (TET) dioxygenases oxidize 5mC into oxidized methylcytosines (oxi-mCs): 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). DNA methylation modifications have multiple functions. For example, 5mC is shown to be associated with diseases and oxi-mC species are reported to have a role in active DNA demethylation through 5mC oxidation and DNA repair, among others, but the detailed mechanisms are poorly understood. Bisulphite sequencing and its various derivatives can be used to gain information about all methylation modifications at single nucleotide resolution. Analysis of bisulphite based sequencing data is complicated due to the convoluted read-outs and experiment-specific variation in biochemistry. Moreover, statistical analysis is often complicated by various confounding effects. How to analyse 5mC and oxi-mC data sets with arbitrary and complex experimental designs is an open and important problem. Results: We propose the first method to quantify oxi-mC species with arbitrary covariate structures from bisulphite based sequencing data. Our probabilistic modeling framework combines a previously proposed hierarchical generative model for oxi-mC-seq data and a general linear model component to account for confounding effects. We show that our method provides accurate methylation level estimates and accurate detection of differential methylation when compared with existing methods. Analysis of novel and published data gave insights into to the demethylation of the forkhead box P3 (Foxp3) locus during the induced T regulatory cell differentiation. We also demonstrate how our covariate model accurately predicts methylation levels of the Foxp3 locus. Collectively, LuxGLM method improves the analysis of DNA methylation modifications, particularly for oxi-mC species. Availability and Implementation: An implementation of the proposed method is available under MIT license at https://github.org/tare/LuxGLM/Peer reviewe

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways

Abstract We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylaseassisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions

An integrative computational systems biology approach identifies differentially regulated dynamic transcriptome signatures which drive the initiation of human T helper cell differentiation

Abstract Background A proper balance between different T helper (Th) cell subsets is necessary for normal functioning of the adaptive immune system. Revealing key genes and pathways driving the differentiation to distinct Th cell lineages provides important insight into underlying molecular mechanisms and new opportunities for modulating the immune response. Previous computational methods to quantify and visualize kinetic differential expression data of three or more lineages to identify reciprocally regulated genes have relied on clustering approaches and regression methods which have time as a factor, but have lacked methods which explicitly model temporal behavior. Results We studied transcriptional dynamics of human umbilical cord blood T helper cells cultured in absence and presence of cytokines promoting Th1 or Th2 differentiation. To identify genes that exhibit distinct lineage commitment dynamics and are specific for initiating differentiation to different Th cell subsets, we developed a novel computational methodology (LIGAP) allowing integrative analysis and visualization of multiple lineages over whole time-course profiles. Applying LIGAP to time-course data from multiple Th cell lineages, we identified and experimentally validated several differentially regulated Th cell subset specific genes as well as reciprocally regulated genes. Combining differentially regulated transcriptional profiles with transcription factor binding site and pathway information, we identified previously known and new putative transcriptional mechanisms involved in Th cell subset differentiation. All differentially regulated genes among the lineages together with an implementation of LIGAP are provided as an open-source resource. Conclusions The LIGAP method is widely applicable to quantify differential time-course dynamics of many types of datasets and generalizes to any number of conditions. It summarizes all the time-course measurements together with the associated uncertainty for visualization and manual assessment purposes. Here we identified novel human Th subset specific transcripts as well as regulatory mechanisms important for the initiation of the Th cell subset differentiation.</p

Dissecting the dynamic changes of 5-hydroxymethylcytosine in T-cell development and differentiation

The discovery of Ten Eleven Translocation proteins, enzymes that oxidize 5-methylcytosine (5mC) in DNA, has revealed novel mechanisms for the regulation of DNA methylation. We have mapped 5-hydroxymethylcytosine (5hmC) at different stages of T-cell development in the thymus and T-cell differentiation in the periphery. We show that 5hmC is enriched in the gene body of highly expressed genes at all developmental stages and that its presence correlates positively with gene expression. Further emphasizing the connection with gene expression, we find that 5hmC is enriched in active thymus-specific enhancers and that genes encoding key transcriptional regulators display high intragenic 5hmC levels in precursor cells at those developmental stages where they exert a positive effect. Our data constitute a valuable resource that will facilitate detailed analysis of the role of 5hmC in T-cell development and differentiation

Le Sacré: Janus philosophique et sociologique

info:eu-repo/semantics/publishe