Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037.
Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2020 Mar 24;117(12):6571-6579. doi: 10.1073/pnas.1921786117. Epub 2020 Mar 10.
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.
MYC 控制着大量长链非编码 RNA（lncRNA）的转录。由于 MYC 是一种普遍存在的癌蛋白，其中一些 lncRNA 可能在癌症中发挥重要作用。我们应用 CRISPR 干扰（CRISPRi）来鉴定 P493-6 和 RAMOS 人淋巴样细胞系中 MYC 驱动细胞增殖所必需的 MYC 调节的 lncRNA。我们确定了 320 个对细胞生长起积极作用的非编码基因座。这些 lncRNA 中的任何一个转录抑制都会降低细胞的增殖能力。通过 RT-qPCR 和带有单个 GFP 表达 sgRNA 构建体的 CRISPRi 竞争测定验证了选定的靶点。我们还通过染色质免疫沉淀显示了 MYC 与两个候选基因启动子的结合。在我们的研究过程中，我们发现 MXD1 蛋白的 SID（SIN3 相互作用域）抑制域在 P493-6 和 RAMOS 细胞中，在文库筛选中耗尽的向导数量和诱导的转录抑制程度方面非常有效。在所使用的细胞系中，SID 优于 KRAB 抑制域，后者通常作为 CRISPRi 中的转录抑制域。SID 转录抑制域与 MS2 适体结合蛋白 MCP 的融合有效，允许构建一个可调节的 doxycycline 调节的 CRISPRi 系统，该系统允许对靶基因进行受控抑制，并将促进促进生长的 lncRNA 的功能分析。
