RNA-Sequencing of LPS-stimulated TLR4 HEK293 cells edited at an immune-response trans-eQTL locus
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ABSTRACT:
PROVIDER: PRJNA607299 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11103100_1.fastq.gz | Fastqsanger.gz | |||
| SRR11103100_2.fastq.gz | Fastqsanger.gz | |||
| SRR11103101_1.fastq.gz | Fastqsanger.gz | |||
| SRR11103101_2.fastq.gz | Fastqsanger.gz | |||
| SRR11103102_1.fastq.gz | Fastqsanger.gz |
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Project description:In order to investigate the genetic effect of an IRF1-associated immune-response trans-eQTL, we used CRISPR editing to introduce deletions at the top variant rs17622517. Five unedited, three heterozygous and five homozygous clones were isolated and RNA-sequencing was performed with no stimulation, with 90min LPS stimulation and 12h LPS stimulation, with two replicates of each. We find a significant correlation between the genetic perturbation here and CRISPRi perturbation of the variant locus, as well as the trans-eQTL effects, suggesting this variant is the causal variant for the immune-response trans-eQTL.
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Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation.
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