RNA sequencing of NF-kB knockout (KO) U937 cells

Transcription factors of NF-kB family play central roles in inflammatory responses. However, most of our current understanding on these factors are based on in vitro context specific studies and animal models, and human specific cellular and pathophysiological functions at molecular level are poorly understood. Here, we have designed a new oligomer based lentiviral pgRNAs-CRISPR-Cas9 knock-out (KO) approach and performed RNA-seq on 11 independent clones representing p50, p52, p65, c-REL and RELB KO cells to systematically study NF-kB family of transcription factors in monocytic cells U937, in steady and inflammatory state. Our analysis reveals factor-specific and common genes and pathways that are dysregulated in an inflammatory state. However, in a steady state respective KO clones showed specific transcriptional perturbations. Incorporating DNA-binding ChIP-ChIP information in the transcriptomic data uncovered enrichment of metabolic and leukemia associated pathways in p65-/- cells. Using acute myeloid leukemia (AML) patient’s data, metabolic, proliferation and xenograft models we showed an anti-leukemic functions of p65 which was mediated by metabolic plasticity and stemness. RNA sequencing was performed on a total of 28 samples representing untreated and LPS treated clones of U937 derived cells; Controls (CTR) (n=3) and NF-kB family of TFs knockout (KO), c-Rel-/- (n=2), p50-/- (n=2), p52-/- (n=2), RELB-/- (n=2), p65-/- (n=3).