![]() Therefore, inhibition of canonical NF-κB signaling can inhibit proliferation or induce apoptosis in a variety of cell- and animal-based cancer models. Second, NEMO is required for the constitutive and chronic activation of canonical NF-κB signaling that occurs in a variety of cancers, and is required for the ability of these cancer cells to proliferate or survive (i.e., avoid apoptosis). First, mutations in the NEMO gene ( IKBKG, chromosome X), which generally compromise the ability of NEMO to support activation of NF-κB, lead to a variety of developmental and immunodeficiency diseases in humans. NEMO is also involved in human disease in two prominent ways. As such, NEMO is a key regulator for activation of the canonical NF-κB pathway by a variety of upstream signals, and NEMO serves to distinguish activation of canonical and non-canonical NF-κB pathways. In contrast, activation of non-canonical processing of NF-κB p100 generally does not require NEMO. In the absence of NEMO, canonical NF-κB signaling cannot be activated. NEMO (NF-κB Essential MOdulator) is a protein that serves as a scaffold for IKKβ in canonical NF-κB signaling. In non-canonical signaling, cytoplasmic NF-κB p100 is phosphorylated by IKKα, an event that induces proteasome-mediated processing of p100 to p52, which then enters the nucleus to affect gene expression. In canonical NF-κB signaling, NF-κB is activated by IKKβ-mediated phosphorylation of the NF-κB inhibitor IκB, which is then degraded to allow NF-κB to enter the nucleus. NF-κB itself is tightly regulated by subcellular localization: that is, NF-κB is located in the cytoplasm when inactive, and is induced to translocate to the nucleus when activated by upstream signals. The mammalian NF-κB transcription factor is involved in the regulation of many cell and organismal processes. In this paper, we have used a CRISPR/Cas9-based targeting approach to investigate cell type-specific promoter expression of a key gene ( NEMO) in NF-κB signaling. Methods for assessing the function of tissue-specific alternative promoter usage for individual genes are limited. For some genes, alternative promoters direct the expression of an identical protein coding region in different cell types or under different conditions by virtue of the promoters being located upstream of distinct 5’ non-translated exons that splice to a common set of downstream coding exons. In many cases, alternative promoters are used for the tissue-specific or developmentally timed expression of a given gene, and abnormal alternative splicing or promoter usage has been associated with human disease, especially cancer. It is estimated that over 50% of human genes have alternative splicing and/or use alternative promoters, and alternative promoter usage has also been coupled to alternative splicing. Much functional gene diversity in humans is generated by the use of alternative splicing and alternative promoters. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. NEMO is a scaffold protein required for canonical NF-κB signaling. The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy.
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