h this study did not identify any PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740540 significant Atf4 sequencing peak in the promoter regions of mouse Nlrp1a, Nlrp1b or Nlrp1c, we decided to re-analyze their ChIP-seq raw data with the publicly available next-generation sequencing analysis software HOMER and confirmed the Atf4 binding motif identified by the nucleotide sequence: TT-A/G-CATCA. Thus, 10 / 16 ATF4 Controls NLRP1 Expression during ER Stress we searched the human NLRP1 promoter region for the presence of an Atf4 consensus site. Interestingly, we found only one match in the middle of the region that we identified as containing a putative ER stress-responsive element. We therefore used a mutagenesis approach to modify three nucleotides of the Atf4 binding motif in the luciferase vector carrying a 2500bp segment from the NLRP1 promoter. When HeLa cells were transfected with the wild-type or ATF4 consensus mutant luciferase vectors, a significant reduction in the luciferase signal was observed in cells transfected with the mutated NLRP1 promoter as compared to cells carrying the wild-type PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740312 promoter following ER stress induction by BFA. To confirm these data, we generated a HeLa 
ATF4 knock-out cell line using CRISPR-Cas9 technology. ER stress-mediated luciferase induction of the 1500bp NLRP1 promoter region was significantly reduced in ATF4-/- HeLa cells compared to wild-type cells. In contrast, the residual luciferase activity induced by ER stress using the 1000bp of NLRP1 promoter was unaffected by loss of ATF4. These data thus identify ATF4 MedChemExpress Elesclomol transcription factor as an activator of NLRP1 gene during ER stress conditions. To extend the analysis of ATF4 effects on NLRP1 expression, we performed ChIP experiments to study stimulation-dependent binding of the ATF4 transcription factor to the human NLRP1 promoter. Consistent with our previous experiments, we observed substantial recruitment of ATF4 to the NLRP1 promoter only upon BFA treatment, while no NLRP1 DNA enrichment was detected in the negative IgG control. Under the same ER stress conditions, we observed ATF4 binding to the ATF3 promoter region, which was used as a positive control. In conclusion, our results demonstrate that upon ER stress the ATF4 transcription factor binds and activates the human NLRP1 promoter. Other transcription factors or other signaling mechanisms, particularly those acting downstream of IRE1, may also contribute to NLRP1 gene expression during ER stress and they are currently under investigation. Discussion Multiple members of the NLR family are capable of forming so-called “inflammasomes”, which are multi-protein complexes that recruit and activate pro-inflammatory caspases and stimulate proteolytic activation of cytokines such as Interleukin-1 and Interleukin-18. Prior studies have recently demonstrated NLRP3 inflammasome activation in response to ER stress conditions. In this study, we present the first evidence that expression of the human NLRP1 gene is transcriptionally up-regulated in response to perturbations of the ER. The human NLRP1 protein is also capable of forming inflammasomes that stimulate caspase-1 activation and processing of pro-IL-1. Interestingly, hereditary polymorphisms in the human NLRP1 gene have been associated with vitiligo, autoimmunity, systemic sclerosis, and increases sensitivity of leprosy. We showed that both the IRE1 and PERK pathways are important for ER stress-induced NLRP1 gene expression. Indeed, we demonstrated that ATF4 transcription factor binds and stimulates t
