Identification of novel regulators of the Nuclear Factor Kappa B pathway in human macrophages
The nuclear factor-kappa B (NF-κB) family of transcription factors has a central role in coordinating the expression of genes that control inflammation, immune responses, cell-proliferation, and a variety of other processes. Ever since its discovery in 1986 in David Baltimore’s lab, the NF-κB pathway has been the prime model of inducible transcription in various cell types, and in response to multiple stimuli. Despite being one of the most well-studied pathway in biology, it still has a lot of unanswered questions associated with it including the events that lead up to its activation in the cytoplasm as well as the sequence of events that lead to the transcription of hundreds of its target genes in the nucleus. Given the pathway’s implication in development and diseases, it has become increasingly important to answer these questions. Here, we present two whole-genome RNAi screens conducted to find novel regulators of this pathway in the physiologically relevant human macrophages in response to Lipopolysaccharides (LPS) and Tumor Necrosis Factor Alpha (TNF). After three levels of screening we have found over 25 potential novel regulators of this pathway, summarized in Chapters 2 and 4. The top hit is the splicing factor and transcriptional co-activator SNW1. We have further validated it as a regulator of the NF-κB pathway in response to multiple stimuli and in five different cell lines (THP-1, U87, 293T, A549, and U2-OS). SNW1 does not seem to affect general constitutive transcription in THP-1 cells but does seem to repress some transcription programs e.g. CREB and NFE2. SNW1 does not regulate the cytoplasmic part of the NF-κB pathway but does complex with the NF-κB hetero dimer in the nucleus on pathway activation. We have shown that it binds to NF-κB’s transcriptional elongation partner p-TEFb and helps recruit it to the NF-κB nuclear complex that contains RNA Polymerase II. We have also shown that SNW1 loses binding from its splicing complex (SNRNP200, SNRNP220) on NF-κB activation. SNW1 is a unique protein shown to be involved in both splicing and transcription and in the case of NF-κB, its role seems to involve recruitment of p-TEFb for effective transcriptional elongation of NF-κB target genes.