CpG site or CpG island, which is important - (Jun/02/2005 )
Oh, One more question before I go.
Hi, PCRman, Can you be more specific? I am really naive on this subject. any literature?
Two papers published last year about siRNA induced methylation silencing.
Morris KV, Chan SW, Jacobsen SE, Looney DJ.
Small interfering RNA-induced transcriptional gene silencing in human cells.
Science. 2004 Aug 27;305(5688):1289-92. Epub 2004 Aug 5.
Kawasaki H, Taira K.
Induction of DNA methylation and gene silencing by short interfering RNAs in human cells.
Nature. 2004 Sep 9;431(7005):211-7. Epub 2004 Aug 15.
Hi pcrman,
You said something that individual CpG sites don't mean anything, just in the context of CpG islands. During my endless frustration with this whole train of scientific thought, I was told to determine the methylation status of individual CpGs (that do NOT qualify as an island) in the promoter region of a gene that we DO know to have reduced mRNA levels in a particular stage of a particular cancer. I discouraged my boss, saying there was no reason to believe site-specific methylation could mean anything. The end result is that there is no significant difference between the two cancer stages we wanted to see, but some significant difference for particular sites between the cancer and the one normal tissue I analyzed. Do you know of any literature that has done anything with site-specific methylation? What if they are CpGs within important transcription factor binding sites? Is there any reason to believe this site-specific methylation could be a factor in transcription levels?
Thanks,
labtechie
p.s. My tenure in my lab is ending and I will be starting medical school... thanks to you and methylnick and everyone else for being my methylation community!
Hey Labtechie,
all the best at medical school, it's a shame to lose another scientist to medicine.
I believe that individual CpG's can affect transcription levels, my manuscript has been bouncing around like a rubber ball because people don't want to believe it.
It's bloody frustrating and I believe it's real!!! I am just going against the majority of published literature that state otherwise that's all
In a recent conference I would say people are starting to become aware of this phenomenon, but as far as I know, there is nothing really out there about this labtechie, only to say that these things are non-functional and useless.
Do drop a line sometime labtechie if you ever want to sway back to research!
Nick
Hi Labtechie and Nick,
Good luck with your new carrer (I know medical school is hard and I was there some time ago). Nick, sorry to hear about your paper.
I am not against the fact that methylation of individual CpG sites in the promoter can impact on transcription. The issue is we know in the genome, most CpGs are methylated except those in the CpG island, ie, even in normal cells, CpGs outside CpG islands are likely methylated. So to study cancer related methylation, I guess better stick to CpG island area. I also think this area is overheated, like the stock market of 2000. Someday, the bubble will burst, we end up gaining nothing, simply because we now know nothing why CpG islands are methylated in cancer.
Thanks for the good wishes, Nick and pcrman! I'm definitely ready for the change...
Basically, I thought I had proven what you said, pcrman, about most CpGs outside of CpG islands being methylated and not showing any kind of change in cancer. However, this promoter does show only a 40-50% methylation (Bisulfite sequencing, 10 clones, from normal human genomic DNA) in certain CpGs, then 80-100% methylation in the cancer (Bisulfite sequencing, 25 clones, cell lines, unfortunately, but it wasn't my choice...I'm just a tech). It's statistically significant, but I don't believe it's earth-shattering. I think my results are just going to be a sentence or two in the paper about characterization of the promoter we are studying. Unlesss you think it could mean something real? Nick I know you are proponent of this theory....
It seems that my question initiate a hot discuss about the role of CpG site and CpG island. Both arguments are educational to me. Hope it’s true to others.
Follow the response, I have two new questions.
1) As suggested by Cyberpostdoc, I submit my sequence to repeatmasker, it masks 63% of my sequence. Fortunately, the major CpG island in Intron I (I mentioned previously) is not masked. But it masks my exon 3, 4, 5 and 6. My gene is only composed of seven exons. The program masks most of the coding region! Is it a usual thing? The cDNA sequence was confirmed, even the protein was crystallized. Why we need to mask all these repeat sequences before perform CpG island or promoter prediction?
2) pcman recommend a specific way to induce methylation using siRNA, but it’s hard to handle. I wonder if there is any chemical can work opposite as Aza-cytindine to methylate DNA in cells. According to my understanding, when cells were treated with Aza-cytindine or other global methylation modifier, their global mRNA expression including housekeeping gene will be changed. So when we want to check the expression change of some genes by QPCR after treatment, what’s for control?
Labtechie,
it's still not clear if a methylation threshold exists whereby 50% island methylation is permissive to transcription while 80-100% is not. I believe a threshold exists from our data, but that's just me. 
Have to get the darn paper accepted! 
CMF, must apologise for hijack the discussion. For your gene, the exons are masked as simple repeats or low complexity? I would be worried if the exons were masked as alu's and line's. You can find this out by the annotation table output by repeatmasked.
There is no methylating agent so to speak, there is a possible invitro methylation route using SssI methylase. But this is in-vitro. As for qPCR the control will be your non-drug treat lines and these are compared to the drug treatment, that's how you would do it, and you can test if the drug affects housekeeping genes by measureing expression before and after drug treatment.
Good luck!
Nick
Good luck!
Nick
Hi, Nick,
I will check the masked sequence later to see if it's Alu or LINE.
When I ask control, I mean the internal gene contol (Say, actin) to quantify the relative gene expression level of my target gene in QPCR. But I wonder global methylation modifier may change the expression level of actin too, which will make the relative expression level unsound. CMF
When I submit the whole gene DNA including introns and exons to repeatmasker , it masked the exon 3, 4, 5 and 6 exons (encoding region)as I mentioned before. But if I only submit the cDNA ORF sequence (exons, around 500bp), it told me no repetitive sequences detected .
I will check the annotation table output (huge) tomorrow and get back to you. I got to go now.