No CpG island=methylation is not important for regulation? - (Oct/25/2005 )

Hi,
I am trying to evaluate the importance of DNA methylation of promoter region on gene transcription during cell differentiation. The problem is that I could not find a CpG island within the promoter and even in the first exon and intron regions. How do your guys think about the importance of DNA methylation on the regulation of gene transcription?
Thanks a lot!

CpG islands are not necessary for controlling transcription for some genes. Even if a CpG island exists, the methylation status (for some genes) does not reflect the transcription status of that particular gene.

Having said this, a CpG island up to 2kb from the start site may exist with your gene of interest. HAve you looked this far up stream?

Nick

Hi qiong,

I was wondering if your promoter region contains known transcription factor binding sites such as SP1 or the like.

You can idedntify transcription factor binding sites with transfac (google transfac), there are a number of transcription factor binding sites that can be methylated and inhibit transcription factor binding leading to inactivation of the gene. Sp1 is the prime example I can think of.

I bellieve transcription factor binding sites do not necessarily need to reside within a CpG island, so if you look for binding sites within your promoter region you may find some and test for methylation status of these by bisulfite seqeuncing!!

good luck

Nick

Nick,
You are right that we have already extensively characterized the promoter of this gene in vitro and in vivo. As you suggested that there are SP1 sites and GC-repressor within the promoter. I appreciate your help. You are really experienced in this field.
I would like to get your help during my bisulfite sequencing experiments since I am really new in this field.

Thanks
Qiong

Good luck Qiong!!

have a browse through all the pinned messages in this forum there is some nice information in them.

I am happy to help Just hope to pass on my experiences to everyone else, as it has been a real nightmare trying to optimise a new technique in the lab from scratch! I am speaking from experience!

Nick

Don't give up Qiong,

I started on the same journey looking at methylation of ZAP-70 which lacks a CpG island. In the end I found my differentially methylated region in intron1.

http://www.haematologica.org/journal/2005/8/1078.html

Providied you are using cell lines then bisulphite sequencing is the best way to ensure you measure every CpG but if you are talking about screening several Kb then I would be tempted to design a pannel of Cobra assays to look at a number of sites across the region first. As for MSP, it's not worth the effort.

In general, i) use a kit for bisulphite treatment and be kind to your treated DNA ii) be realistic in your amplicon size (<300bp) iii) make sure that your primers contain no CpGs and no polymorphisms

Finally, don't forget that the sense and antisense strands of DNA look very different after bisulphite treatment and so you can design primers for either strand to get your region of interest amplified and analysed.

Thanks, Autopark
Thanks for your suggestion. I am trying my bisulfite treatment now. Hope it work!!!

Qiong