Project description:We have performed a epitranscriptomics study in which we first tretaed TNF-alpha in HeLa cells and DMSO was used as a negative control. Total RNA was isolated from TNF-alpha treated cells as well as control cells and apoptotic rate was determined by flow cytometry. Total RNAs were subjected to miCLIP to identify differentially m6A methylated RNAs. We then transcriptionally analyzed apoptotic genes which have differential m6A methylation in TNF-alpha tretament by qPCR. Afterthat, candidate genes were examined at the level of translation by polysome fractioantion assay.
Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
Project description:Toxoplasma gondii (T. gondii) is an opportunistic parasite. After infection, macrophages finely regulate the immune response to restrict parasite proliferation. It is well-known that N6-methyladenosine (m6A) plays a critical role in fine-tuning gene expression. To investigate whether m6A modification is involved in regulating the anti-infection immune response in macrophages against T. gondii, this study utilized T. gondii tachyzoites from the RH strain to infect human THP-1 macrophages. qPCR and ELISA results showed that T. gondii infection mounted the expression of TNF-α. RNA-seq profiling showed that T. gondii infection was associated with difference in genes from pathway associated with TNF signaling. Expression of m6A regulators were evaluated using qPCR and Western blotting. T. gondii infection increased the abundance of m6A methyltransferase WTAP and demethylase FTO. Joint analysis of RNA-seq and m6A-seq data was utilized for enriching differentially expressed genes with significantly altered m6A modifications. After T. gondii infection, the m6A levels of genes associated with TNF signaling were significantly altered. In this study, we found that m6A methylation involved in T. gondii infection induced TNF-α expression.
Project description:Anti-TNF (tumor necrosis factor) monoclonal antibodies have revolutionized management of Inflammatory bowel disease. Their common features include high efficacy but also immunogenicity and increased infection risk. Since 2013, two generics or biosimilars of the first anti-TNF have been registered in Europe, which long lerm safety profile needs yet to be established.
This prospective, multicenter, observational cohort study will assess safety of treatment of anti-TNF monoclonal antibodies in inflammatory bowel disease patients in Poland.
Eligible are consecutive patients in whom anti-TNF is started for Crohn’s disease, ulcerative colitis or indeterminate colitis between January 1st, 2014 and December 31st, 2015. Data to be collected include demography, Montreal classification, indication to treatment, previous treatment, operations, extraintestinal manifestations and concomitant diseases. Data on response, tolerability and safety of anti-TNF and on concomitant treatment will be collected. Adverse events logs will be completed. Majority of IBD centres in Poland, pediatric and adult, academic and regional, have agreed to participate in the study.
As a result of the study, the frequency of adverse events in a cohort of Polish IBD patients on various anti-TNFs will be established.
Project description:The epigenetic marking of DNA, histones, and RNA is central for regulating gene expression in development and disease. On DNA, well-characterized 5-methylcytosine (5mC) controls the chromatin state. On mRNA, the most prevalent internal mark, N6-methyladenosine (m6A), installed by METTL3-METTL14 methyltransferases, regulates RNA metabolism. Recent evidences link RNA modifications with epigenetics, notably by connecting m6A to histone marks. The crosstalk between m6A and DNA methylation, however, remains poorly understood. Here we reveal a direct mechanistic link between m6A and DNA methylation. Our findings indicate a proximal co-occurrence of m6A and 5mC marks at gene body and this contributes to enhanced gene expression. We show that the m6A writer METTL3 regulates intragenic methylation and gene expression. Mechanistically, we evidence a direct binding of METTL3-METTL14 to the 5mC methyltransferase DNMT1 and reveal that chromatin-tethered METTL14 recruits DNMT1 to chromatin. We demonstrate that gene-body 5mC and mRNA-stability-enhancing effect of coding-sequence m6A contribute to increased gene expression. We further substantiate our findings in embryonic stem cells by showing that METTL3 regulates gene-body methylation and expression of key differentiation markers. Altogether, our work establishes a key layer of gene expression regulation, involving a direct mechanistic crosstalk between DNA methylation and RNA methylation machinery.
Project description:Epigenetic modifications are known to profoundly affect the development and behavior of social insects. In the well-known caste differentiation process of honeybee (Apis mellifera), female larvae with identical genomes are fed royal jellydifferently and develop into either normal workers or into very large, long-lived, and extremely fecund queens, and the queen-worker asymmetry of honeybee is known to be result largely to differential genomic imprinting during larval development that involves DNA methylation-based regulation. The discovery of reversible N6-methyladenosine (m6A) RNA methylation modification has defined a new era for RNA-metabolism-related genetic regulation, yet much remains unknown about m6A-mediated post-transcriptional regulatory mechanisms. Here, we report the first honeybee RNA m6A methylome. Specifically, we used the m6A-seq technique to examine the RNA m6A methylomes of honeybee larvae, including queen and worker larvae at multiple instar stages. We identified multiple conserved features of m6A methylation machinery and transcriptome-wide m6A distribution trends among insect species, and observed that m6A marks exert functions in regulating caste differentiation, with apparently particularly strong functional impacts on fifth instar worker larvae. Functional annotation of differentially methylated candidate caste-differentiation-related transcripts revealed many known regulators of caste differentiation (e.g. ILP-2, p110, PI3K, and JHAMT etc.) as well as the widely-studied Vitellogenin gene, which has not previously been implicated in caste differentiation. As ever-more regulatory roles for m6A marks are discovered, honeybees may become an excellent model studying the biology of such epi-transcriptomic regulatory systems, from embryonic development through holometabolous caste-specific development and on towards behavior and the emergent social hierarchies underlying eusociality in animals.
Project description:N6-methyl-adenosine (m6A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m6A by mapping the m6A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m6A modification, including transcripts encoding core pluripotency transcription factors. m6A is enriched over 3M-bM-^@M-^Y untranslated regions at defined sequence motifs, and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m6A methylases, led to m6A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESCM-bM-^@M-^Ys exit from self-renewal towards differentiation into several lineages in vitro and in vivo. Thus, m6A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages. Examing m6A modification differences in two different cell types
Project description:Endothelial-mesenchymal-transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which are known to facilitate tumor progression. We have previously shown that EndMT is present in pancreatic tumors and that deficiency of the Tie1 receptor induces EndMT in human endothelial cells. Pancreatic tumors are characterized by the presence of tumor necrosis factor-α (TNF-α). We now show that TNF-α strongly induces human endothelial cells to undergo EndMT. In order to know the secretory feature of cells which undergo EndMT by TNF-α, we conducted a comparative analysis of HMVEC secretome treated or not for 24h and 48h with TNF-α. Secretome study shows that cells treated with TNF-α have an important fibroblast-like secretory capacity, and a proinflamatory signature. Moreover, Ingenuity Pathway Analysis (IPA) shows that pathways implicated in migration, inflammation and fibrosis are predicted to be activated and that necrosis and apoptosis pathways are inhibited. Accordingly cell survival, viability and cycle progression are activated. We show that TNF-α- treated cells secrete proteins related to 16 protumoral pathways, confirming their fibroblastic characteristic. Finally, among the predicted upstream regulators activated, IPA analysis shows that, TNFSF12 and its receptor are present at hight levels in PDAC patients. Altogether these results show the fibroblastic characteristic of treated cells and demonstrate that TNF-α induces CAFs.
Project description:The epigenetic marking of DNA, histones, and RNA is central for regulating gene expression in development and disease. On DNA, well-characterized 5-methylcytosine (5mC) controls the chromatin state. On mRNA, the most prevalent internal mark, N6-methyladenosine (m6A), installed by METTL3-METTL14 methyltransferases, regulates RNA metabolism. Recent evidences link RNA modifications with epigenetics, notably by connecting m6A to histone marks. The crosstalk between m6A and DNA methylation, however, remains poorly understood. Here we reveal a direct mechanistic link between m6A and DNA methylation. Our findings indicate a proximal co-occurrence of m6A and 5mC marks at gene body and this contributes to enhanced gene expression. We show that the m6A writer METTL3 regulates intragenic methylation and gene expression. Mechanistically, we evidence a direct binding of METTL3-METTL14 to the 5mC methyltransferase DNMT1 and reveal that chromatin-tethered METTL14 recruits DNMT1 to chromatin. We demonstrate that gene-body 5mC and mRNA-stability-enhancing effect of coding-sequence m6A contribute to increased gene expression. We further substantiate our findings in embryonic stem cells by showing that METTL3 regulates gene-body methylation and expression of key differentiation markers. Altogether, our work establishes a key layer of gene expression regulation, involving a direct mechanistic crosstalk between DNA methylation and RNA methylation machinery.
Project description:Blood (mRNA and miRNA) and skin mRNA transcriptomes were investigated across three time-points in a pilot investigation of ten severe psoriasis patients, treated with the tumor necrosis factor (TNF) inhibitor, etanercept. We used illumina RNA-sequencing to analyse the mRNA transcriptome in blood