High throughput screens identify long non-coding RNA LOUP contributions to NFkB signaling and monocyte differentation.

Long non-coding RNAs (lncRNAs) account for the largest portion of RNA from the transcriptome and yet most of their functions remain unknown. Here we performed a high throughput CRISPRi reporter-based screen to identify lncRNAs that regulate TLR4-NFkB signaling in human monocytes and those that contribute to PMA-induced differentiation . We successfully identified numerous non-coding and protein-coding genes that can positively or negatively regulate these pathways. To understand the functional roles of lncRNAs in TLR4/NFkB signaling, we chose to further study one top candidate from our screens, LOUP (lncRNA originating from upstream regulatory element of SPI1 [also known as PU.1]. It’s been previously shown by Trinh et al. that the LOUP transcript directly mediates interactions between a nearby enhancer element of SPI1 and the SPI1 promoter. SPI1 is a transcription factor that drives myeloid cell fate, is highly expressed in monocytes, and regulates transcription of inflammatory genes. Here we’ve demonstrated that while LOUP expression can enhance SPI1 expression in monocytes, knockdown of LOUP also leads to a broad upregulation of NFkB targeted genes, both at baseline and upon TLR4-NFkB activation, and that this mechanism is independent of LOUP’s regulatory effects on SPI1. We found that LOUP harbors three small open reading frames (sORFs) capable of being translated, and that two sORFs in particular, are responsible for LOUP’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. For TLR4-NFkB screening, library infected and selected THP1-NFKB-dCas9 were stimulated with 200 ng/ml of LPS for 24 h to induce expression of GFP (NF-kB responsive) across three clonal lines: A, B, and C. Prior to sorting, cells were collected in FACS buffer (1XPBS, 1%FBS, 5mM EDTA). Stimulated cells were analyzed by flow cytometry alongside unstimulated cells to ensure the mean fluorescence intensity (MFI) of stimulated cells was > 10-fold compared to unstimulated cells. All flow cytometry experiments and screening were conducted on a BD FACSAria II. GFP was excited using a 488-nm laser and detected using a 525/50-nm filter. Sorting was conducted using 4-way purity into 2 tubes and a 100-mm nozzle. Cells were gated by forward (FSC-A) and side scatter (SSC-A) for live cells, then for single cells using FSC-A/FSC-H. Lastly, we evaluated GFP expression (SSC versus GFP), FACS sorted and collected the top/bottom 20% into separate tubes. At least 100 cells/sgRNA (100X coverage) for each sorted population were collected and cryo-preserved in 90% FBS, 10% DMSO for later processing. DNA was extracted and sgRNA guide sequences were sequenced and counted. Unsorted +LPS samples were collected for each clonal line as well (A,B,C), thus each clonal line has GFP_Hi, GFP_Lo, and Unsorted frations For PMA differentiation screening, the same library was infected and selected THP1-NFKB-dCas9 were stimulated with 2nM PMA on day 8 and 5nM PMA on day 9 to induce differentiation. On day 11 differentiated macrophages were collected and DNA was extracted and sgRNAs sequences were sequenced and counted. Comparisons were made against those present at Day 3 without PMA treatment. The same clonal lines (A,B,C) were used and all samples were used in the analysis with the exception of Clone B +PMA Day 11 because cell coverage fell below 500X during screening.