Side-effects of 5-aza - non-specific demethylation in primary culture (May/04/2005 )
I've been demethylating primary cultures of astrocytes with varying doses of 5-aza-cytidine, up to 20uM, for 7 days. My gene of interest increases in mRNA expression, but I've found through bisulfite sequencing that this is probably not due to demethylation of its specific promoter.
I'm looking into other specific genes whose activation may affect my protein's expression, but does anyone know if there are any common nonspecific effects of 5-aza treatment, other than general toxicity? Such as housekeeping genes that are generally activated, etc. I'm trying to explain why my gene is increased in expression by 5-aza treatment, although its promoter is hypomethylated normally. Thanks.
Is your gene of interest part of a biochemical pathway? if so, that would be the first place to start to look.
If you know of any transcription factors that bind to the promoter region, the expression of these factors would affect the transcription of your gene of interest.
From my experience 5-aza-C also decondensates pereicentromeric chromatin, and you could extraopolate this to say that the heterochromatin is affected. It may be worth your while to look at the chromatin environment where your gene of interest lies and see if this has change from a state that is non-permissive to transcription to a permissive state.
Okay, there are several problems here which need to be addressed.
First, if you want to look for genes that are activated by AZA look at early time periods.
Find a time point where AZA first demethylates a known target gene and use this time point.
Any time point beyond that and you will undoubtedly be looking at secondary effects of a cell that is responding to a highly toxic substance. For example, if you look at some array profiles you will see a large overlap between AZA and TSA activity NOT because they are doing similar biochemical things but because they are TOXIC.
Treating cells with AZA for 7 days seems excessive. Read papers. In the literature it is rare to treat with AZA for more than 3 days.
When treating with AZA, the cell shuts down and transcription of many genes is terminated due to toxicity. Therefore , if your gene is NOT turned off by AZA it may automatically seem to be more highly expressed because there is simply less RNA in the cell and your RNA of interest will end up being a larger proportion of the total amount. More simply, Total amount of RNA prior to AZA -100 (arbitrary number). Total of your RNA before AZA = 1. Therefore it is 1% of total.
After AZA total RNA in cell = 50 (due to gene inactivation in toxic environment). Your RNA still =1.
BUT the total percentage will double to 2% due to less total RNA.
It is difficult to get real results with long periods of AZA treatments.