How short can be the CpG island? - methylation (Jun/12/2004 )
Is anyone know how short of the CpG island can be? let say if the CG sequence of about 30bp, is that possible the short CpG sequence be methylated?
Please read this thread http://www.protocol-online.org/forums/inde...?showtopic=3160
In my opinion, a stretch of DNA of 30 bp in length with high CpG content cannot be defined as a CpG island. The original CpG island criteria for length is at least 200 bp, later Jones group further extended the criteria to 500 bp.
It may be methylated. If you are going to map this area, be cautioned that the accepted concept is, in normal cells, CpG sites outside CpG islands are usually methylated. That means if you want to study the methylation status in cancer cells for example, the significance of this area will be questionable.
[quote=pcrman,Jun 12 2004, 11:45 PM]
It may be methylated. If you are going to map this area, be cautioned that the accepted concept is, in normal cells, CpG sites outside CpG islands are usually methylated. That means if you want to study the methylation status in cancer cells for example, the significance of this area will be questionable. [/quote]
Thanks for your advice! As you said
"in normal cells, CpG sites outside CpG islands are usually methylated." Is that possible that the high CpG site (about 30bp) right at the begining of CDS of a gene can be methylated and affected the expression of that proteins, i.e silencing the protein expression? The reason I asked is that I have cloned a gene with a high CpG site (about 30bp) right at the begining of CDS. After selection under G418, I can found some clones with high RNA level of that gene, but no protein expression of that gene. This is why I am wondering is that possible the high CpG site at the begining of CDS would be methylated and caused no expression of protein.
The other question is what if there are some high CpG sites within the CDS of the gene, would that also be methylated as well, if so, is that mean the protein will be truncated and/or with abnormal function??
Thank you so much for your advice in advance!
Our understanding toward how individual methylation of individual CpG sites contributes to transcriptional silencing is still incomplete. We see in the literature that people do methylation analysis without first identifying the presence of CpG island within the region studied. The methylation of the high density CpG region at the beginning of your gene could affect your gene transcription but not TRANSLATION. You said you noticed high mRNA expression but not protein expression. This is not a typical result of methylation mediated gene silencing which only occurs at transcriptional level.
CpG islands can extend from promoter into coding region, therefore methylation of any of those CpG sites may have impact on transcription. As to which CpG sites are critical, you have to decide experimentally. You can treat your cells with 5'-aza-cytidine, a demethylating agent, and examine, before and after treatment, mRNA expression and methylation changes. If methylated CpG sites become unmethylated after treatment with restoration of mRNA expression, then you can say that the methylation of those CpG sites is responsible for transcriptional silencing of related gene.
Hope that helps.