The acetyltransferase p300 is recruited in trans to multiple enhancer sites by lncSmad7. [ChIRP-seq]
Ontology highlight
ABSTRACT: The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear. We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.
Project description:The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear. We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.
Project description:The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear. We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.
Project description:The histone acetyltransferase p300 (also known as KAT3B) is a general transcriptional coactivator that introduces the H3K27ac mark on enhancers triggering their activation and gene transcription. Genome-wide screenings demonstrated that a large fraction of long non-coding RNAs (lncRNAs) plays a role in cellular processes and organ development although the underlying molecular mechanisms remain largely unclear. We found 122 lncRNAs that interacts directly with p300. In depth analysis of one of these, lncSmad7, is required to maintain ESC self-renewal and it interacts to the C-terminal domain of p300. lncSmad7 also contains predicted RNA-DNA Hoogsteen forming base pairing. Combined Chromatin Isolation by RNA precipitation followed by sequencing (ChIRP-seq) together with CRISPR/Cas9 mutagenesis of the target sites demonstrate that lncSmad7 binds and recruits p300 to enhancers in trans, to trigger enhancer acetylation and transcriptional activation of its target genes. Thus, these results unveil a new mechanism by which p300 is recruited to the genome.
Project description:While thousands of long non-coding RNAs (lncRNAs) are expressed in higher eukaryotes, the potential regulatory roles of lncRNAs in regulated gene transcription programs remain rather poorly understood. Here, we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 and PCGEM1, bind successively to the androgen receptor (AR) and strongly enhance both ligand-dependent and ligand-independent AR-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the C-terminally acetylated AR on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM, to the N-terminally methylated AR. Unexpectedly, recognition of the H3K4me3 promoter mark by the PHD finger-domain of Pygopus2, recruited by PCGEM1, proves to enhance selective looping of AR-bound enhancers to target gene promoters in these cells, revealing a novel aspect of ligand-induced enhancer-promoter interactions. In M-bM-^@M-^\resistantM-bM-^@M-^] prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for the robust activation of both truncated and full length AR, causing DHT-independent activation of the AR transcriptional program and cell proliferation. Conditionally-expressed short hairpin RNA (shRNA)-mediated targeting of these lncRNAs in these resistant cancer cell lines strongly suppressed xenograft growth in vivo. Together, these results suggest that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumors. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq); ChIRP-seq data for a lincRNA (PCGEM1). LNCaP cells were grown to 30-50% confluence and siRNA/ASO transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturerM-bM-^@M-^Ys instructions. Control samples were transfected with scramble ASO and control siRNA, respectively. On the following day of transfection, the cells were cultured in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM for 1 hour we have sequenced the DNA sequences that are associated with the presence of a RNA molecule on a genome wide scale. PCGEM1 probe, -DHT PCGEM1 probe, +DHT
Project description:Accumulating evidence highlights the role of long non-coding RNAs (lncRNA) in cellular homeostasis, and their dysregulation in disease settings. Most lncRNAs function by interacting with proteins or protein complexes. While several orthogonal methods have been developed to identify these proteins, each method has its inherent strengths and limitations. Here, we combine two RNA-centric methods ChIRP-MS and RNA-BioID to obtain a comprehensive list of proteins that interact with the well-known lncRNA HOTAIR. Overexpression of HOTAIR has been associated with a metastasis-promoting phenotype in various cancers. Although HOTAIR is known to bind with PRC2 and LSD1 protein complexes, an unbiased and comprehensive method to map its interactome has not yet been performed. Both ChIRP-MS and RNA-BioID data sets show an association of HOTAIR with mitoribosomes, suggesting HOTAIR has functions independent of its (post-)transcriptional mode-of-action.
Project description:Chromatin-associated long non-coding RNAs (ca-lncRNAs) play important roles in transcriptional regulation. To comprehensively investigate the chromatin interactome of an enhancer-derived ca-lncRNA, LEENE, we performed the Chromatin isolation by RNA purification (ChIRP) to pull down LEENE-associated DNAs, which were subjected to DNA-seq. Specifically, we used 10 previously validated tiling nucleotides targeting different domains based on the predicted secondary structure. LEENE was overexpressed by adenovirus to enhance the ChIRP-seq signals.
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:The spatial co-presence of aberrant long non-coding RNAs (lncRNAs) and abnormal coding genes contributes to malignancy development in various tumors. However, precise coordinated mechanisms underlying this phenomenon in tumorigenesis remains incompletely understood. Here, we show that Prohibitin 2 (PHB2) orchestrates the transcription of an oncogenic CASC15-New-Isoform 2 (CANT2) lncRNA and the coding tumor-suppressor gene CCBE1, thereby accelerating melanoma tumorigenesis. In melanoma cells, to further capture the binding proteins of CANT2, ChIRP-MS was performed.