An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation

Abstract

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.

Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model

“…Considering the increased risk of off-target of multi-gRNAs, other optimization strategies need to be tested to improve CRISPRi effect on active genes in the future. Recently, KRAB combination with MeCP2 or LSD1 or alternative repressors like SIN3-interacting domain (SID) have been reported to achieve superior efficiency [ 38 , 40 , 42 ] and are worth further testing at active genes in different cell types.…”

CRISPR-based technology to silence the expression of IncRNAs

“…Large Cas9 fusion proteins can be challenging to express using lentiviral vectors and could be toxic to some cells due to their interaction with corepressors. By contrast, Raffeiner et al (7) only require the stable expression of dCas9, which should be functionally inert in the absence of an sgRNA. The MCP-SID fusion protein is relatively small and thus can be readily expressed using a lentiviral vector that coexpresses an sgRNA, enabling efficient inducible gene repression.…”

“…Indeed, technologies that partially knock down genes may better model the effect of drugs, which rarely completely inactivate their targets in vivo. And lastly, as demonstrated by Raffeiner et al (7), CRISPRi technologies are effective tools to probe the noncoding genome. From these perspectives, it will be important to start building a public database of CRISPRi results so that metaanalyses can be performed, which can highlight cell type-and context-specific effects of lncRNAs.…”

“…The authors (7) hypothesize that their approach might have been more successful if they had used a repression domain that was highly active in MYCtransformed lymphomas. MYC target gene activation is normally opposed by complexes that include MXD1 (MAD), which competes with MYC for binding to MAX and recruits the transcriptional corepressor SIN3A (15).…”

“…All this technological wizardly is in the service of identifying functional important lncRNAs that may play a role in MYC-driven B cell lymphomas. Importantly, the screen performed by Raffeiner et al (7) rediscovers the importance of MIR17HG, which is a MYCregulated lncRNA that encodes several microRNAs that target the tumor suppressor PTEN (17). Among the novel lncRNAs that are essential in BL cells, Raffeiner et al validate two-SNHG17 and SNHG26-as bona fide MYC target genes using chromatin immunoprecipitation assays.…”