CRISPRi screens identify the lncRNA, LOUP , as a multifunctional locus regulating macrophage differentiation and inflammatory signaling

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 121; no. 22; p. e2322524121
• Main Authors: Halasz, Haley, Malekos, Eric, Covarrubias, Sergio, Yitiz, Samira, Montano, Christy, Sudek, Lisa, Katzman, Sol, Liu, S John, Horlbeck, Max A, Namvar, Leila, Weissman, Jonathan S, Carpenter, Susan
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 28.05.2024
• Subjects: Biological Sciences; Cell Differentiation - genetics; CRISPR-Cas Systems; Differentiation; Gene Expression Regulation; Genes; High-throughput screening; Humans; Immune system; Inflammation; Inflammation - genetics; Inflammation - metabolism; Innate immunity; Macrophages; Macrophages - cytology; Macrophages - metabolism; Monocytes; Monocytes - cytology; Monocytes - metabolism; NF-kappa B - metabolism; NF-κB protein; Non-coding RNA; Open reading frames; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; PU.1 protein; RNA, Long Noncoding - genetics; RNA, Long Noncoding - metabolism; Signal Transduction; TLR4 protein; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - metabolism; Toll-like receptors; Trans-Activators - genetics; Trans-Activators - metabolism; Transcriptomes; long noncoding RNA; macrophage; CRISPRi; inflammation; short encoded peptide
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2322524121

Long noncoding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome, yet most of their functions remain unknown. Here, we performed two independent high-throughput CRISPRi screens to understand the role of lncRNAs in monocyte function and differentiation. The first was a reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and the second screen identified lncRNAs involved in monocyte to macrophage differentiation. We successfully identified numerous noncoding and protein-coding genes that can positively or negatively regulate inflammation and differentiation. To understand the functional roles of lncRNAs in both processes, we chose to further study the lncRNA [lncRNA originating from upstream regulatory element of (also known as PU.1)], as it emerged as a top hit in both screens. Not only does regulate its neighboring gene, the myeloid fate-determining factor , thereby affecting monocyte to macrophage differentiation, but knockdown of leads to a broad upregulation of NFkB-targeted genes at baseline and upon TLR4-NFkB activation. also harbors three small open reading frames capable of being translated and are responsible for 's ability to negatively regulate TLR4/NFkB signaling. This work emphasizes the value of high-throughput screening to rapidly identify functional lncRNAs in the innate immune system.