question after getting BSP result - (Nov/14/2005 )

Hi,
I am trying to determine whether DNA demethylation is involved in cell specific gene activiation during differentiation. With the help of Nick, I did get the sequencing results back. The results showed that one repressive cis-element containing one CG (which was characterized as GC-repressor in our field) is methylated in differentiated condition whereas the CG is not methylated in undifferentiated condition (Moreover, this GC-repressor was demonstrated to be bound by Sp1/Sp3 in our lab). Other CG (which are not involved in cis-element) are not methylated in differentiaed condition compared to the methylated status in undifferentiated condition, which is consistent with my original hypothesis that DNA is demethylated during the differentiation of my cell type.
Here, I am trying to ask:
1. Is this result a possible artifact of the assay itself?
2. What should I do next since I plan to methylate the Luc-reporter by SssI at the begining (Now the result showed that there exist different status of methylation in a short distance)?

I really apprepriate any help from you because our lab has no any experience in this field.

Thanks

Qiong

highly unlikely,

does the GC repressor reside in a gene and if so are you able to test for expression of this gene?

A reporter assay will be the way to go and give you clues to the functionality of this repressor.

is it possible to antagonise the methylation with a drug say 5-aza-deoxycytidine and reverse the differentiation? another way would be to use RNAi to reduce the levels of DNMT activity and look for a reversal of pheotype.

The invtro assay will be good to perform, but as most reviewers these days are looking for function, you have to demonstrate this somehow.

Nick

Thanks, Nick
That is why I am thinking it is an artifact. And I am trying to include more cell types and in vivo tissues.

Thanks
Qiong