Do transfected vectors bind histones and/or methylate? - (Feb/08/2006 )
I mean, does it make sense to transfect cells with, for example, a reporter vector with luciferase and treat cells with HDACs inhibitors? As well, does it make sense to transfect a reporter vector and treat cells with demethylating agents without methylating it previously in vitro?
thanks a lot
i suppose it makes sense if your vector is silenced upon transfection.
if your vector was made in a methylation deficeinct bacteria, then it won't be methylated by the bacteria and you will isolate the vector in an unmethylated form.
you can not demethylate DNA in-vitro, there is no known demethylase enzyme to date. all the agents used require active DNA replication invivo and replace the cytosine with a form of cytosine that can not be methylated.
hi nick, thanks for your answer!
so then, if the bacteria are not methylation deficient, should i suppose that all the cytosines in my vector are methylated?
yes, check the strain of bacteria you are using, and see if it's methylation deficient.
Another quick test would be to digest your isolated plasmid with a methylation senstive restriction enzyme, this will tell you right away if your vector has been methylated or not.
thanks again, nick.
so, my bacteria are not methylation deficient (so the vector is methylated) and the promoter cloned in the reporter vector is regulated by methylation. since a transiently transfected vector does not replicate in the cell (am i right on this?), methylation status of the vector won´t change if i treat cells with AZAdc after transfection. is this right?
...and another question: if i make my vector in methylation deficient bacteria and i transfect it, will it be de novo methylated in the cell? does the de novo methylation require dna replication as the demethylation?
thanks a lot
azadC treatment of cells is a whole new ball game, and to perform this on a transiently transfected vector, well it's pretty tricky I would have to say.
be easier to have the vector in a methylation deficient bacteria and go from there.
DNA and the vector can be denovo methylated by the cell. I think this will happen only in long term culture if at all. If you are only short term culturing, it shouldn't affect you too much, furthermore I take it your vector has some form of selection on it, this would also help as the vector is needed for cell survival under selction.
thank you very much, nick!
As far as association of transiently transfected plasmids with histones go then the answer is 'yes' they do associate with histones to some degree (through nothing compared to stable transfectants).
There are plenty of papers available online which detail the use of HDACs, especially sodium butyrate and Trichostatin A, to increase the expresion of transiently transfected reporters. In my previous research I investigated the activity of the MDR1 promoter. This can be upregulated by both NaBu and TsA only 24-48 hours following transfection.
Look up Scotto, Jin, Hu or Minuzzo for recent applications and Frommel or Morrow for the original work on the promoter in the early 90s. Can be found on PubMed
thanks for your reply doc!
but is there direct evidence of actual binding? i mean, could this induction of reporter plasmids be due to upregulation/activation of some endogenous transcription factors after tsa/NaB treatment?