Plasmid CpG methylation after transfection - (Nov/16/2006 )

I am thinking of an experiment looking at the initiation of methylation of a promoter of my gene of interest. My hypothesis is that is some cell lines there maybe differences in the ability of the promtoer to be methylation. This maybe dependent on the differenitation stage of the cell. I have MSP primer specific for my plasmid (ie will not pick up endogenous copies of the gene). I was wondering if anyone has used a technique like this to address eukaryotic methylation?

Have a look at this paper:


1: Oncogene. 2002 Feb 7;21(7):1048-61.

Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in
prostate cancer cells.

Song JZ, Stirzaker C, Harrison J, Melki JR, Clark SJ.

Kanematsu Laboratories, Sydney Cancer Centre, Royal Prince Alfred Hospital,
Camperdown, NSW 2050, Australia.

Understanding what triggers hypermethylation of tumour suppressor genes in
cancer cells is critical if we are to discern the role of methylation in the
oncogenic process. CpG sites in CpG island promoters, that span most tumour
suppressor genes, remain unmethylated in the normal cell, despite the fact that
CpG sites are the prime target for de novo methylation by the DNA
methyltransferases. The CpG island-associated with the GSTP1 gene is an
intriguing example of a CpG rich region which is susceptible to hypermethylation
in the majority of prostate tumours and yet is unmethylated in the normal
prostate cell. In this study we evaluate a number of factors purported to be
involved in hypermethylation to test their role in triggering hypermethylation
of GSTP1 in prostate cancer DU145 and LNCaP cells. We find that hypermethylation
is not associated with (1) elevated expression of the DNA methyltranferases, or
(2) removal of Sp1 transcription factor binding sites in the CpG island or (3)
removal of CpG island boundary elements or (4) prior gene silencing. Instead our
results support a model that requires a combination of prior gene silencing and
random "seeds" of methylation to trigger hypermethylation of the GSTP1 gene in
the prostate cancer cell. We propose that the GSTP1 gene is initially silenced
in the prostate cancer and random sites of methylation accumulate that result in
subsequent hypermethylation and chromatin remodelling.

Thanks a bunch. That was the kind of study I was thinking about.