49 research outputs found
The long intergenic non-coding RNA CCR492 functions as a let-7 competitive endogenous RNA to regulate c-Myc expression
3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells
Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches
Intragenic DNA methylation prevents spurious transcription initiation.
In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cance
Genome-wide analysis identifies a functional association of Tet1 and Polycomb repressive complex 2 in mouse embryonic stem cells
BACKGROUND: Ten-Eleven Translocation (TETs)proteins mediate the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet1 is expressed at high levels in mouse embryonic stem cells (ESCs), where it mediates the induction of 5hmC decoration on gene-regulatory elements. While the function of Tet1 is known, the mechanisms of its specificity remain unclear. RESULTS: We perform a genome-wide comparative analysis of 5hmC in pluripotent ESCs, as well as in differentiated embryonic and adult cells. We find that 5hmC co-localization with Polycomb repressive complex 2 (PRC2) is specific to ESCs and is absent in differentiated cells. Tet1 in ESCs is distributed on bivalent genes in two independent pools: one with Sin3a centered at non-hydroxymethylated transcription start sites and another centered downstream from these sites. This latter pool of Tet1 co-localizes with 5hmC and PRC2. Through co-immunoprecipitation experiments, we show that Tet1 forms a complex with PRC2 specifically in ESCs. Genome-wide analysis of 5hmC profiles in ESCs following knockdown of the PRC2 subunit Suz12 shows a reduction of 5hmC within promoter sequences, specifically at H3K27me3-positive regions of bivalent promoters. CONCLUSIONS: In ESCs, PRC2 recruits Tet1 to chromatin at H3K27me3 positive regions of the genome, with 5hmC enriched in a broad peak centered 455 bp after the transcription start site and dependent on the PRC2 component Suz12. These results suggest that PRC2-dependent recruitment of Tet1 contributes to epigenetic plasticity throughout cell differentiation
High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues
Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification