Next Generation Sequencing to check the enrichment of RIPK1/BAF on chromatin, RIPK1-dependent chromatin state and RIPK1-dependent transcriptome during inflammatory responses.

RIPK1 is a master regulator of multiple cell death pathways, including apoptosis and necroptosis, and inflammation. Multiple RIPK1 inhibitors have been advanced into human clinical trials as new therapeutics for human inflammatory and neurodegenerative diseases, including ALS and AD. However, while the mechanism of cytosolic RIPK1 in control of cell death has been extensively investigated, how the activation of RIPK1 may promote transcription of proinflammatory cytokines is unclear as a nuclear function of RIPK1 has not been explored, nor is it clear if and how RIPK1 kinase activity may directly mediate inflammation independent of cell death. Here we show that nuclear RIPK1 is recruited by specific transcription factors and binds to the BAF complex on active enhancers and promoters marked by H3K4me1 and H3K27ac. Nuclear RIPK1 mediates the phosphorylation of SMARCC2, a key component of the BAF complex, to promote chromatin remodeling and the transcription of specific proinflammatory genes. Our results suggest that RIPK1 kinase serves a transcriptional coregulator in nucleus that can transmit extracellular stimuli to BAF complex to modulate the chromatin accessibility and directly regulate the transcription of specific genes involved in mediating inflammatory responses. Overall design: Chromatin acessibility and transcriptiome in RIPK1D325A/D325A; RIPK3-/- MEFs were analyzed. p-S166-RIPK1, SMARCC2, BRG1 enrichiment on chromatin in RIPK1D325A/D325A; RIPK3-/- MEFs treated with TNF for 2h, 4h were analyzed. p-S166-RIPK1, SMARCC2, BRG1 enrichiment on chromatin in spinal cords from ALS mouse model and human patients were analyzed.