De novo plasmid methylation in eukaryotic cells - (Feb/19/2007 )

Hi everybody,

I'm trying to do a luciferase assay with methylated/unmethylated reporter constructs + cotransfection with MBDs in mammalian cells. I seem to get repression of reporter gene activity also (to some extent) when the reporter construct is not methylated. I thought of two things: 1) binding of the MBDs also to unmethylated CpGs, 2) spontaneous de novo methylation of the reporter construct. Does anybody know of any previous publications about these thigs? Any help is appreciated .

Josepha

Hi Josepha,

two explanations come directly to my mind: a) overepression of MDBs could lead to a general increase of Histonedeacetylase activity, which could lead to a rather unspecific repression of the reporter gene and b) hemi-methylated DNA, to which MDBs can also bind (but as far as I know to a lesser degree, what would be in accordance with your results) but which is not visible in every methylation analysis (as long as you are not BSPing both strands).
Spontaneous de novo methylation seems rather unlikely to me. Which MDBs are you using? If you have MeCP amongst them, I would rather believe in spontaneous de-methylation, still very unlikely!

But maybe somebody has a better explanation?

Krümel

spontaneous methylation does occur in cell lines and it won't be surprising you are seeing it in your reporter construct. It is part of the host defense mechanism whereby any foreign DNA is hypermethylated and inactivated.

How long after transfection are you performing the reporter assay? maybe reduce that time to detect the transient expression of your construct.

Also as Krumel said, MBDs are known to bind unmethylated DNA for sure, look up any of Peter Jones and Paul Wade's papers (that's PA Jones and MBD) and there is something I recall from their recent papers.

Nick

I think this paper from the Wade group is the one you were looking for, Josepha (PubmedID: 12788925).
De novo methylation - okay, Nick is right here, not sooooooo unlikely, but really a question of timing. There are some reports of sequences especially prone to de novo methylation - maybe you check those? Or, to exclude this factor, you can try using the DNMT3a/DNMT3b null ES cell line (described in 16439359).

Krümel