I am finding a massive down-regulation of XBP1-dependent activity in cerebellum homogenates in one of my experiments. The activity of 37 of the 43 genes in my data set that are directly regulated by XBP1 are decreased. I understand that XBP1 is related to ER stress and is also involved in MHCII folding. However, I am fairly certain that my treatment is causing an increase in oxidative stress in the brain. I would expect XBP1-dependent activity would go up to clean up the mess associated with oxidative damage. So why would XBP1-regulated activity be turned off?
I am using the Affy Mouse ST 1.0 gene expression array, CARMAweb for the fold change/p value analysis, and IPA for pathway/upstream regulator analysis.
Hi Michael, I also see a disconnect between XBP1 and oxidative stress in my work. I did a siRNA library screen and found that silencing of a single protein can lead to a significant decrease in XBP1 splicing but a significant increase in the ARE oxidative stress reporter. It may be that the oxidative stress is not turning on ER stress as you expect. When I treat with an ER stress agent such as thap, I do not see oxidative stress and when I treat with an oxidative stress agent such as DTT, I do not see ER stress. My system is fibroblasts though, so it may be cell type specific. It also may be that other parts of UPR are turned on, such as ATF6 or PERK, rather than IRE1, to upregulate chaperone expression and turn on ERAD to deal with misfolded protein. You may want to check genes that are specifically regulated by ATF6 as well as ERAD genes. Hope this helps :)
In my hands, oxidative stress increases unspliced levels of XBP-1, which is stable in C. elegans. Unspliced XBP-1 still has DNA binding affinity, but no activation domain. So, in theory, unspliced XBP-1 could outcompete functional XBP-1S at the site of transcription, thus hindering gene activation. This work will soon be published, so let me know if you would like more information. I hope this helps.
Thanks. I have whole genome data so I will look at the genes you suggest and see what's up. I am meeting with some genomics people this afternoon so maybe that'll help.
Jody, I looked at the ER Stress Pathway using IPA (pathway analysis software) and most of the genes are downregulated. ATF6, BiP, PERK, EIF2S1, and TRAF2 are all inhibited. There doesn't seem to be a significant difference in IRE1, though.
Kira, I wish I knew if our array was specific for the unspliced or spliced version. I suspect it is the unspliced, so it probably includes spliced. But the gene itself is down-regulated (or, at least, there's less mRNA), as well as all downstream activity. IF what you are saying is true, wouldn't you still have more spliced XBP1 mRNA around?
I'm finding all sorts of papers that talk about what happens when there is MORE of XBP1 around, but almost nothing about what it means when it's shut off. Haha. Guess I'll keep digging. Thanks for the suggestions.
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