Global examination of LncMyoD binding sites using ChIRP-seq
Ontology highlight
ABSTRACT: We report the application of ChIRP-seq on examination of LncMyoD binding sittes in differentiated myotubes We observed the global binding sites of LncMyoD in differentiated myotubes.
Project description:We report the application of ChIPmentation on examination of H3K27ac histone mark in cultured satellite cells with siRNA treatment targeting LncMyoD. We observed no difference of H3K27ac mark around transcription start sites (TSS) after LncMyoD KD.
Project description:Increasing evidence suggests that Long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of novel intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding-protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well-conserved between human and mouse, its locus, gene structure and function is preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. In order to perform an unbiased search for downstream signaling pathways perturbed by LncMyodD downregulation, microarrays were performed on myoblasts treated with control vs LncMyoD shRNAs. Total RNA was extracted using the TRIzol reagent (Invitrogen) and purified with Qiagen RNeasy separation columns (Qiagen) from myoblasts treated with control vs. LncMyoD shRNA. First-strand cDNA was synthesized and hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix).
Project description:To understand the effect of LncMyod knockdown on cultured satellite cells, we isolated satellite cells from mouse hindlimb and cultured them in vitro with siRNA treatment targeting LncMyod. Following RNA-seq showed that loss of LncMyod leads to difficiencies in myogenic differentiation.
Project description:Increasing evidence suggests that Long non-coding RNAs (LncRNAs) represent a new class of regulators of stem cells. However, the roles of LncRNAs in stem cell maintenance and myogenesis remain largely unexamined. For this study, hundreds of novel intergenic LncRNAs were identified that are expressed in myoblasts and regulated during differentiation. One of these LncRNAs, termed LncMyoD, is encoded next to the Myod gene and is directly activated by MyoD during myoblast differentiation. Knockdown of LncMyoD strongly inhibits terminal muscle differentiation largely due to a failure to exit the cell cycle. LncMyoD directly binds to IGF2-mRNA-binding-protein 2 (IMP2) and negatively regulates IMP2-mediated translation of proliferation genes such as N-Ras and c-Myc. While the RNA sequence of LncMyoD is not well-conserved between human and mouse, its locus, gene structure and function is preserved. The MyoD-LncMyoD-IMP2 pathway elucidates a mechanism as to how MyoD blocks proliferation to create a permissive state for differentiation. In order to perform an unbiased search for downstream signaling pathways perturbed by LncMyodD downregulation, microarrays were performed on myoblasts treated with control vs LncMyoD shRNAs.
Project description:In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes as well as RNA polymerase II (Pol II) binding sites in mouse fetal myotubes. Examination of Sox6 and Pol II binding sites in mouse fetal primary myotubes
Project description:We employed ChIRP in combination with proteomic strategy to systematically discover HOTAIR-interacting proteins. Three independent biological replicates of ChIRP-MS experiment were performed, alongside negative controls.
Project description:We identified orthologs of the roX lncRNAs across diverse Drosophilid species, and then mapped the genomic binding sites of roX1 and roX2 in four Drosophila species (D. melanogaster, D. willistoni, D. virilis, and D. busckii) using ChIRP-seq (chromatin isolation by RNA Purification and sequencing), thus revealing the interplay of the evolution of roX1 and roX2 and their genomic binding sites.
Project description:In this study, we profiled for LINE1 binding loci in RSeT+DT naïve hES cells with the Chromatin Isolation by RNA Purification (ChIRP)-seq strategy. We followed a previously published ChIRP protocol described (Percharde et al. 2018; Lu et al. 2020).