Similar Datasets
Project description:MYC controls the transcription of large numbers of long noncoding RNAs (lncRNAs). Since MYC is a ubiquitous oncoprotein, some of these lncRNAs probably play a significant role in cancer. We applied CRISPR interference (CRISPRi) to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 noncoding loci that play positive roles in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RT-qPCR and in CRISPRi competition assays with individual GFP-expressing sgRNA constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID (SIN3-interacting domain) derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, SID is superior to the KRAB repressor domain, which serves routinely as a transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP, allowing the construction of a doxycycline-regulatable CRISPRi system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
| S-EPMC7104257 | biostudies-literature
Project description:MYC controls the transcription of large numbers of long non-coding RNAs. Since MYC is a ubiquitous oncoprotein, some of these long non-coding RNAs (lncRNAs) probably play a significant role in cancer. We applied CRISPRi to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 non-coding loci that play a positive role in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RTqPCR and in CRISPRi-competition assays with individual GFP-expressing sgRNA-constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, it is superior to the KRAB repressor domain which serves routinely as transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP allowing the construction of a doxycycline regulatable CRISPRi-system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
2020-04-06 | GSE141491 | GEO
Project description:MYC controls the transcription of large numbers of long non-coding RNAs. Since MYC is a ubiquitous oncoprotein, some of these long non-coding RNAs (lncRNAs) probably play a significant role in cancer. We applied CRISPRi to the identification of MYC-regulated lncRNAs that are required for MYC-driven cell proliferation in the P493-6 and RAMOS human lymphoid cell lines. We identified 320 non-coding loci that play a positive role in cell growth. Transcriptional repression of any one of these lncRNAs reduces the proliferative capacity of the cells. Selected hits were validated by RTqPCR and in CRISPRi-competition assays with individual GFP-expressing sgRNA-constructs. We also showed binding of MYC to the promoter of two candidate genes by chromatin immunoprecipitation. In the course of our studies, we discovered that the repressor domain SID derived from the MXD1 protein is highly effective in P493-6 and RAMOS cells in terms of the number of guides depleted in library screening and the extent of the induced transcriptional repression. In the cell lines used, it is superior to the KRAB repressor domain which serves routinely as transcriptional repressor domain in CRISPRi. The SID transcriptional repressor domain is effective as a fusion to the MS2 aptamer binding protein MCP allowing the construction of a doxycycline regulatable CRISPRi-system that allows controlled repression of targeted genes and will facilitate the functional analysis of growth-promoting lncRNAs.
2020-04-06 | GSE141482 | GEO
Project description:An MXD1-derived repressor peptide identifies non-coding mediators of MYC-driven cell proliferation
| PRJNA594049 | ENA
Project description:An MXD1-derived repressor peptide identifies non-coding mediators of MYC-driven cell proliferation [RNA-seq]
| PRJNA593587 | ENA
Project description:An MXD1-derived repressor peptide identifies non-coding mediators of MYC-driven cell proliferation [Amplicon-seq]
| PRJNA594048 | ENA
Project description:Using RNA-seq (RNA sequencing) of ribosome-depleted RNA, we have identified 1,273 lncRNAs (long non-coding RNAs) in P493-6 human B-cells. Of these, 534 are either up- or downregulated in response to MYC overexpression. An increase in MYC occupancy near their TSS (transcription start sites) was observed for MYC-responsive lncRNAs suggesting these are direct MYC targets. MYC binds to the same TSS across several cell lines, but the number of TSS bound depends on cellular MYC levels and increases with higher MYC concentrations. Despite this concordance in promoter binding, a majority of expressed lncRNAs are specific for one cell line, suggesting a determinant role of additional, possibly differentiation-specific factors in the activity of MYC-bound lncRNA promoters. A significant fraction of the lncRNA transcripts lack polyadenylation. The RNA-seq data were confirmed on eight selected lncRNAs by NRO (nuclear run-on) and RT-qPCR (quantitative reverse transcription PCR).
| S-EPMC4350361 | biostudies-literature
Project description:Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the main oncogenic driver in lung cancer that can be activated by gene mutation or amplification, but whether long non-coding RNAs (lncRNAs) regulate its activation remains unknown. Through gain and loss of function approaches, we identified that lncRNA HIF1A-As2, a KRAS-induced lncRNA, is required for cell proliferation, epithelial-mesenchymal transition (EMT) and tumor propagation in non-small cell lung cancer (NSCLC) in vitro and in vivo. Integrative analysis of HIF1A-As2 transcriptomic profiling reveals that HIF1A-As2 modulates gene expression in trans, particularly regulating transcriptional factor genes including MYC. Mechanistically, HIF1A-As2 epigenetically activates MYC by recruiting DHX9 on MYC promoter, consequently stimulating the transcription of MYC and its target genes. In addition, KRAS promotes HIF1A-As2 expression via the induction of MYC, suggesting HIF1A-As2 and MYC form a double-regulatory loop to strengthen cell proliferation and tumor metastasis in lung cancer. Inhibition of HIF1A-As2 by LNA GapmeR antisense oligonucleotides (ASO) significantly improves sensitization to 10058-F4 (a MYC-specific inhibitor) and cisplatin treatment in PDX and KRASLSLG12D-driven lung tumors, respectively.
| S-EPMC10089381 | biostudies-literature
Project description:Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborates strongly with c-Myc in lymphomagenesis. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. We show here that Gfi1 augmented the expression of c-Myc protein in cells transfected with c-Myc expression constructs. The N-terminal SNAG domain and C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon Gfi1 overexpression, but reduced following Gfi1 knockdown or knockout, which was associated with a decline in the expression of c-Myc-activated target genes. Consistent with its role in the regulation of c-Myc expression, Gfi1 promoted Myc-driven cell cycle progression and proliferation. Together, these data reveal a novel mechanism by which Gfi1 augments the biological function of c-Myc and may have implications for understanding the functional collaboration between Gfi1 and c-Myc in lymphomagenesis.
| S-EPMC7554040 | biostudies-literature
Project description:MYC oncogene family members are broadly implicated in human cancers, yet are considered "undruggable" as they encode transcription factors. MYC also carries out essential functions in proliferative tissues, suggesting that its inhibition could cause severe side effects. We elected to identify synthetic lethal interactions with c-MYC overexpression (MYC-SL) in a collection of ~3,300 druggable genes, using high-throughput siRNA screening. Of 49 genes selected for follow-up, 48 were confirmed by independent retesting and approximately one-third selectively induced accumulation of DNA damage, consistent with enrichment in DNA-repair genes by functional annotation. In addition, genes involved in histone acetylation and transcriptional elongation, such as TRRAP and BRD4, were identified, indicating that the screen revealed known MYC-associated pathways. For in vivo validation we selected CSNK1e, a kinase whose expression correlated with MYCN amplification in neuroblastoma (an established MYC-driven cancer). Using RNAi and available small-molecule inhibitors, we confirmed that inhibition of CSNK1e halted growth of MYCN-amplified neuroblastoma xenografts. CSNK1e had previously been implicated in the regulation of developmental pathways and circadian rhythms, whereas our data provide a previously unknown link with oncogenic MYC. Furthermore, expression of CSNK1e correlated with c-MYC and its transcriptional signature in other human cancers, indicating potential broad therapeutic implications of targeting CSNK1e function. In summary, through a functional genomics approach, pathways essential in the context of oncogenic MYC but not to normal cells were identified, thus revealing a rich therapeutic space linked to a previously "undruggable" oncogene.
| S-EPMC3386069 | biostudies-literature