how to rigorously demonstrate the one gene is up-regulated in the transcriptiona - (Aug/24/2006 )
I found the level of a protein increases a lot in response to radiation. I also found that the luciferase activity driven by the promoter region of the gene significantly increased under the same condition. I am not sure whether this is enough to say that the up-regulation of the protein is regulated in the transcriptional level, because one of my committee members questioned that I did not show rigorously that the upregulation is regulated in the transcriptional level. Does that mean I have to do RT-PCR or Real-Time PCR to show the endogenous transcription activity increase?
ps: what else can induce the protein level increase besides the gene transcription increase?
Thanks a lot.
Increased expression at protein level and even at mRNA level as detected by RT-PCR or Northern doesn't necessarily mean increased transcription. Changes in mRNA stability and translation affect mRNA and protein levels. The rigorous method to demonstrate upregulated transcription is nuclear run on assay which is technically challenging.
Thanks for the reply. Because I am designing experiments to test whether radiation will induce more AP-1 to bind to the promoter region, which may upregulate the gene expression. I was suggested that I should not proceed with further experiments unless I rigoroulsy prove that the gene is regulated in the transcriptional level. So, any suggestions in case it is not regulated in the transcriptional level? I mean, any methods I can use to explain the protein level increase?
Besides nuclear run on assay, there exist other alternatives to establish a causal-effect relationship between radiation and transcriptional activation of your gene via increased AP-1 binding.
You can use ChIP assay to pull down AP-1 associated with the promoter to see if radiation enriched AP-1.
You can use ChIP assay to pull down other active transcriptional components such as poly II, acetylated histones to prove that radiation does induce an actively transcribed promoter.
In addition to Western, you definitely need to assess mRNA levels by RT-PCR or real-time RT-PCR.
If you can provide data that there is an increase in mRNA level, and the promoter is activated (ChIP data), you should be able to make a good case without doing the formidable nuclear run on assay.
It seems to me that an increase in protein level observed under a particular condition without a corresponding increase in promoter activity might indicate regulation at some other level (translation, mRNA stability), but the fact that you also see a corresponding increase in promoter activity under the same conditions indicates that the difference you see is, in fact, due at least in part to upregulation at the transcript level.
I mean, it might not be the only thing going on (you could have an increase in transcription and an increase in mRNA half-life and an increase in translation all working at once), but the contribution of any these other things, if applicable, does not negate your finding that the transcription level is increased under the condition tested relative to controls.
I suppose you could do a northern blot to show directly there is more specific transcript in a given amount of treated vs untreated cells...
Thanks for the nice suggestion. I will do RT PCR first to see what will happen. Hopefully I can get some positive results.
Just to add to the list of possible other factors, don't forget riboswitches in the 5' or 3' UTR. But the RTPCR is your first stop.