Project description:Naïve T cells of Mettl3 KO and WT were pulse labeled by s4U for 15 min before and after IL-7 stimulation for 15min/30min/45min/60min/75min/90min. The total input RNAs or s4U nascent RNAs were isolated and prepared for next-generation sequencing. The RNA response to IL-7 and RNA decay rates were then modeled and calculated, to infer the m6A/Mettl3 effects.
Project description:Naïve T cells were obtained by StemCell CD4+ T cell isolation kit of spleen from Mettl3 KO and WT mice followed by FACS sorting (CD4+CD25-CD45RB-Hi). Raw sequencing reads were aligned to the mouse genome (mm10) with Tophat, and gene expression levels were measured by Cufflinks.
Project description:Next-generation sequencing measuring ribosome mRNA occupancy and translation efficiency in CD4+ T cells isolated from Mettl3 KO and WT mice
Project description:In this study we identify Mettl3, an m6A RNA modification writer, as a critical regulator for terminating naïve pluripotency and a positive maintainer of primed pluripotency in vitro and in vivo. Remarkably, Mettl3 knockout pre-implantation epiblasts and naïve ES cells, entirely lack m6A on coding mRNAs and are viable. Yet, they fail to adequately terminate the naïve pluripotent state, and subsequently undergo aberrant priming and early lineage commitment at the post-implantation stage. A comprehensive functional and genomic analysis involving profiling of m6A, RNA transcription and translation in Mettl3 wild-type and knockout pluripotent and differentiated cells, identified m6A as a critical determinant that destabilizes secondary naïve specific pluripotency genes Esrrb, Klf4 and Nanog, and restrains their transcript stability and translation efficiency. In summary, our findings provide for the first time evidence for a critical role for an mRNA epigenetic modification in early mammalian development in vivo, and identify a mechanism that functionally regulates mouse naïve and primed pluripotency in an opposing manner. Ribosome footprint (Ribo-Seq) was measured from mouse embryonic stem cells and mouse embriod bodies, in WT and Mettl3-KO cell lines.
Project description:In this study we identify Mettl3, an m6A RNA modification writer, as a critical regulator for terminating naïve pluripotency and a positive maintainer of primed pluripotency in vitro and in vivo. Remarkably, Mettl3 knockout pre-implantation epiblasts and naïve ES cells, entirely lack m6A on coding mRNAs and are viable. Yet, they fail to adequately terminate the naïve pluripotent state, and subsequently undergo aberrant priming and early lineage commitment at the post-implantation stage. A comprehensive functional and genomic analysis involving profiling of m6A, RNA transcription and translation in Mettl3 wild-type and knockout pluripotent and differentiated cells, identified m6A as a critical determinant that destabilizes secondary naïve specific pluripotency genes Esrrb, Klf4 and Nanog, and restrains their transcript stability and translation efficiency. In summary, our findings provide for the first time evidence for a critical role for an mRNA epigenetic modification in early mammalian development in vivo, and identify a mechanism that functionally regulates mouse naïve and primed pluripotency in an opposing manner. polyA RNA-seq was measured in mouse embryonic stem cells (ESCs) and embroid bodies (EBs), each in WT and in Mettl3-KO cell lines. RNA-seq was measured also from WT mouse embronic fibroblasts (MEF). 3 biological replicates are available from ESCs and 2 from EBs. Replicate C in ESCs was measured alongside protein levels (SILAC) and was used for the analysis of that assay.
Project description:Genome-wide DNA methylation profiling of HCT116 WT, HCT116 DNMT1 and DNMT3B double KO, and breast cancer tumors by next generation Infinium assay
Project description:Gene expression data from wild-type and Bcl6-/- naive CD4 T cells In order to find genes regulated by Bcl6 in follicular helper T cells Naïve CD4 T cells were sorted from wild-type (WT) and T cell-specific conditional Bcl6-/- (KO) mice-- 8 samples, 4 WT and 4 KO
Project description:Gene expression data from wild-type and Bcl6-/- naive CD4 T cells In order to find genes regulated by Bcl6 in follicular helper T cells Naïve CD4 T cells were sorted from wild-type (WT) and T cell-specific conditional Bcl6-/- (KO) mice-- 8 samples, 4 WT and 4 KO
