CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system
CRISPR screening identifies M1AP as a new MYC regulator with a promoter-reporter system
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DOI：
10.7717/peerj.9046
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发表时间：
2020-05-06
期刊：
影响因子：
2.7
通讯作者：
Kitagawa, Masanobu
中科院分区：
文献类型：
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作者：
Yamamoto, Akiko;Kurata, Morito;Kitagawa, Masanobu
背景MYC是导致许多人类癌症中肿瘤发生的原癌基因之一。虽然MYC调控的机制还不完全清楚，但了解控制MYC转录活性的综合机制将导致治疗靶点。CRISPR/Cas9文库系统是一种简单而强大的筛选技术。本研究旨在使用具有新启动子-报告系统的CRISPR激活文库鉴定MYC的新转录上游激活因子。MYC启动子-报告子系统是用可光转化的荧光蛋白Dendra 2开发的，并命名为“pMYC-启动子-Dendra 2”。“这种MYC启动子-报告基因系统被设计为在（3.1 kb）处具有近端MYC启动子。将CRISPR活化文库和pMYC-启动子-Dendra 2两者诱导至HEK 293 T细胞，并且通过细胞分选仪单独收集Dendra 2阳性细胞，其被认为MYC应该被上调。在收集的169个细胞中，成功建立了12个克隆。将pMYC-启动子-Dendra 2再次转染到这12个克隆中，12个克隆中有2个显示Dendra 2阳性。在该过程中，通过利用Dendra 2的光开关特性，正确区分具有非特异性自发荧光的细胞。使用这两个Dendra 2阳性克隆的提取的基因组DNA，进行聚合酶链式反应（PCR）以扩增含有指导RNA（gRNA）的区域，其由CRISPR活化文库引入。最终，鉴定了PLEKHO 2、MICU、MBTPS 1和M1AP，并使用CRISPR激活系统将这些gRNA再次单独转染到HEK 293 T细胞中。仅M1AP gRNA转染的细胞显示Dendra 2阳性荧光。然后，通过实时定量PCR和western blot，用强力霉素诱导型载体的M1AP过表达载体证实M1AP诱导高MYC表达。此外，双荧光素酶测定显示启动子活性显著增加，并且MYC mRNA在M1AP过表达细胞中更高。M1AP在多种肿瘤中均呈高表达，但仅在急性髓系白血病中与MYC呈正相关。本研究证实，使用CRISPR文库技术的实验方法有效地用于鉴定激活内源性MYC的分子。这种方法将有助于阐明MYC表达的调控机制，以及支持进一步的药物研究，对恶性肿瘤。
Background. MYC is one of the proto-oncogenes contributing to tumorigenesis in many human cancers. Although the mechanism of MYC regulation is still not fully understood, learning about the comprehensive mechanism controlling the transcriptional activity of MYC will lead to therapeutic targets. The CRISPR/Cas9 library system is a simple and powerful screening technique. This study aims to identify new transcriptional upstream activators of MYC using the CRISPR activation library with new promoter-reporter systems.Methods and Results. The MYC promoter-reporter system was developed with a photoconvertible fluorescent protein, Dendra2, and named "pMYC-promoter-Dendra2." This MYC promoter-reporter system was designed to harbor a proximal MYC promoter at (3.1 kb). Both the CRISPR activation library and pMYC-promoter-Dendra2 were induced to HEK 293T cells, and Dendra2-positive cells, that are supposed that MYC should be upregulated, were collected individually by a cell sorter. Among the 169 cells collected, 12 clones were successfully established. Then, pMYC-promoter-Dendra2 was transfected again into these 12 clones, and two of 12 clones showed Dendra2 positivity. In this procedure, the cells with non-specific autofluorescence were correctly distinguished by utilizing the photoswitchable character of Dendra2. Using extracted genomic DNA of these two Dendra2 positive clones, polymerase chain reaction (PCR) was performed to amplify the guide RNA (gRNA) containing region, which was introduced by the CRISPR activation library. Eventually, PLEKHO2, MICU, MBTPS1, and M1AP were identified, and these gRNAs were transfected individually into HEK 293T cells again using the CRISPR activation system. Only M1AP gRNA transfected cells showed Dendra2-positive fluorescence. Then, the overexpression vector for M1AP with a doxycycline-inducible vector confirmed that M1AP induced high MYC expression by real-time quantitative PCR and western blot. Furthermore, the dual-luciferase assay showed a significant increase of promoter activity, and MYC mRNA was higher in M1AP- overexpressing cells. M1AP is highly expressed in several cancers, though, a positive correlation between M1AP and MYC was observed only in human acute myeloid leukemia.Conclusion. The present study confirmed that the experimental method using the CRISPR library technology functions effectively for the identification of molecules that activate endogenous MYC. This method will help elucidate the regulatory mechanism of MYC expression, as well as supporting further drug research against malignant tumors.