Project description:Recently, the repertoire of human small nucleolar noncoding RNAs (snoRNAs) and their potential functions has expanded with the discovery of new snoRNAs and messenger RNA (mRNA) targets, for which snoRNA-guided modifications may influence their stability, translatability, and splicing. We previously identified snoRNAs that are abundant in healthy human muscle progenitor cells. In this study, we demonstrated that SNORA40 and SNORA70 loss-of-function impairs myogenic differentiation. Interestingly, gain-of-function can rescue impaired differentiation muscle progenitor cells in myotonic dystrophy type 1 (DM1). We identified cyclin D3 (CCND3) mRNA, which is partially located in the nucleolus, as a target for SNORA40 and SNORA70, which are required for its pseudouridylated status. Expression of the CCND3 protein is required for muscle progenitors to exit the cell-cycle when they are induced to differentiate. We revealed that this switch requires SNORA40/70. Finally, we observed that DM1 cells show reduced levels of SNORA40/70 and undetectable CCND3 protein. However, restoring normal levels of SNORA40/70 partially restored CCND3 protein expression, coinciding with improved cell fusion capacity in DM1 muscle progenitors. Collectively, these data suggest that this effect may stem from SNORA40/70-dependent pseudouridylation of CCND3 mRNA, emphasizing snoRNAs as key players in normal and pathological muscle differentiation.
Project description:Recently, the repertoire of human small nucleolar ncRNAs (snoRNAs) and their potential functions have been expanded by the discovery of new snoRNAs and mRNA targets, for which snoRNA-guided modifications may influence their stability, translatability, or splicing. We previously identified snoRNAs that are abundant in healthy human muscle progenitors. Here, we showed that SNORA40 and SNORA70 loss-of-function impairs myogenic differentiation. Strikingly, their gain-of-function can rescue the impaired differentiation muscle progenitor cells in Myotonic Dystrophy type 1 (DM1). We identified the CCND3 mRNA, partly located in the nucleolus, as a target of SNORA40 and SNORA70, which are needed for its pseudouridylated status. Expression of the CCND3 protein is required for muscle progenitors to exit the cell cycle when they are induced to differentiation. Here, we revealed that this switch requires SNORA40/70. Finally, we showed that rescuing reduced levels found in DM1 cells restores the expression of CCND3 through pseudouridylation of its mRNA, and ultimately resumes the cell fusion capacity of DM1 muscle progenitors.
Project description:We performed CTCF ChIP-seq to determine the extent of CTCF occupancy alterations in two DM1 patient lymphoblastoid cell lines (LCLs) compared to an unaffected control LCL. Our results show that there were no large-scale changes in CTCF occupancy in the DM1 patient cells either genome-wide or in a 2 Mb region centered around the expanded repeats of DMPK.
Project description:DM1-iPSCs were generated from the peripheral blood of male adult patients. All clones were generated using episomal vectors. For skeletal muscle differentiation of the DM1-iPSCs, tet-on MYOD1 expression vector was transfected into DM1-iPSCs. To examine whether DM1-iPSC clones with different repeat sizes have acquired different characteristics, we isolated DM1-iPSC clones with different repeat sizes. We isolated 2 clones (S45 and S118) showing repeat size of 400~900, 2 clones (S14 and S24) showing repeat size of 1200~1600, and 2 clones (S1 and S16) showing repeat size of 1800~2800.
Project description:We are investigating the transcriptional response of changes in RNA steady-state levels between normal and DM1. We used microarrays to detail the global programme of gene expression differences in normal or DM1 myoblasts. Keywords: comparison Two types of cells were analyzed, normal or DM1 deficient. The expression differences were compared to each other and we have deciphered a gene expression profile that is representative of DM1 deficiency.
Project description:Ado-trastuzumab emtansine(T-DM1) is manufactured by conjugating DM1 to lysine of Trastuzumab via a non-cleavable linker in a non-specific mode.conjugated site of T-DM1 were identified using a bottom-up strategy through LC-HR-MS/MS.
Project description:The esophageal cancer cell line OE-19 was used to select resistant cells to T-DM1. They were selected by prolonged exposure to increasing concentrations of T-DM1 in the absence or presence of ciclosporin A (CsA). Two cell lines resulted from these experimental conditions: OE-19 TR in the absence of CsA and OE-19 TCR in the presence of CsA. The parental cell line was named OE-19 S. The present study aims to detect the transcriptomic changes between OE-19 S, OE-19 TR and OE-19 TCR cell lines to identify the deregulated pathways involved in acquired resistance to T-DM1.
Project description:The mechanisms of resistance to the antibody-drug conjugate, T-DM1, were studied on clones derived from breast cancer cell line, BT474. Microarray analyses were performed to explore potential transcriptomic differences among the distinct cell lines.
Project description:We performed DNA sequencing of potential biallelic SNPs in HD-B and DM1-A patient cell lines. These potential biallelic SNPs were identified in the 4C-seq interaction data. We selected a subset of these SNPs for confirmation by PCR, so we amplified the genomic regions that contained these potential SNPs and performed 2 x 150 bp paired-end sequencing on Illumina MiSeq nano.