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An MXD1-derived repressor peptide identifies non-coding mediators of MYC-driven cell proliferation [Amplicon-seq]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141491 | GEO | 2020/04/06

REPOSITORIES: GEO

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An MXD1-derived repressor peptide identifies non-coding mediators of MYC-driven cell proliferation [RNA-seq]

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.

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Gene expression analysis to identify Myc-regulated long noncoding RNAs in B cells with variable Myc levels

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