Analysing methylation of plant promoter and enchancer - (Aug/28/2013 )
I was wondering if anyone out there can answer these questions for me.
I am currently analysing silencing in my transgene promoter in rice. Methylation restriciton digests suggest that my promoter (553bp) is methylated and so is the UBI enchancer gene (1050bp) that follows the promoter (the construct is - Promoter-Enhancer-Transgene-Terminator). We would like to carry out some Bisulfite sequencing to confrim that they are methylated and I am currently designing BS Primers. According to MethPrimer I may have a CpG Island (411bp) that spans the end of my promoter and into the beginning of my enchancer gene. So my questions are:
1) Should I be interested in seqencing only the overlapping CpG Island? As I understand it only methylation of the Islands has biological significance, or should I be trying to amplify the entire promoter.
2) If I bisulfite sequence the CpG Island should I amplify only a region that is within the promoter? Most of the programs that generate BSP only give me primers for the region of CpG Island within the enchancer gene.
Apologies for the very basic questions but I've very new to epigenetics and no-one in my lab studies methylation.
Thanks in advance
You can start with the CpG island region for sequencing. If you see something interesting, you can then expand into neighboring regions.
If I bisulfite sequence the CpG Island should I amplify only a region that is within the promoter? Most of the programs that generate BSP only give me primers for the region of CpG Island within the enchancer gene.
Methylation in enhancer sequence could also has biological implication and deserves investigation. If you separate promoter and enhancer sequences when designing primers, you can have primers designed for both regions.
Since you are working with plants, many existing primer design programs such as methprimer deal with mammalian methylation which supposedly only occurs to cytosine within CpGs. You should keep that in mind when using those programs.
it is worth to try very different annealing temperatures; for instance BS-PCRs in rat and mice are usually a whopping 10 degrees celsius lower than in humans in my PCR machines.
Determining CpG islands is tricky business, sliding windows are used in the reference genome, and it depends where you start when and where CpG islands are called. In methprimer they use much smaller definition than in UCSC for instance, so methprimer may call regions "CpG" islands, but these may not exist in reference genome annotations.
Do not only focus on CpG islands as CpG poor regions are more likely to have functional regulatory methylation, there is a evolutionary pressure on CpGs, so if they are still there they are more likely to be functional. Only when studying tissue differences and cancer CpG islands rule. (in my humble experience/opinion)
best of luck my plant loving friend
and enhancers are coool! DNA methylation is often low when TFs bind and several TF known to bind enhancers are actually DNA methylation sensitive proteins! Binding if or if there is no methylation!
Thank you Epigenetist and Enthusiast for answering my questions.
I have noted that there is alot of variability between different programs for detecting CpGs in my sequence and (I'm very ashamed to say) I also didn't consider the fact that most of these programs are based upon methylation in mammals.
I think I'm going to try to amplify both the promoter and, if I get time, the enchancer gene. I've been finding it very difficult to amplify regions of the the promoter from Bisulfite-treated DNA because the sequence contains lots of cytosines and adenines. But I've ordered new primers designed using online programs like Methprimer and hopfully playing around with the annealing tempreature will do the job.
Thanks again you've both been a great help
I know that in plants methylation can occur to cytosines in any sequence context. Have you taken that into consideration when designing your primers?
I have noted that there is alot of variability between different programs for detecting CpGs in my sequence
Do you mean detecting cpG islands? Don't worry much about the different results from different programs. It just give you a sense which regions are CpG rich.
That's very true, I hadn't considered that at all! I am such a bad plant geneticist.
I just did a quick google search and found a really great program called Kismeth that can design BS primers for use with plants (it can also analyse your bisulfite sequences like Cymate). Looking at the primers the program has suggested against my DNA sequence it looks like they do take the probability that any cystosine can be methylated into account. I have ordered the primers so fingers crossed they'll work. I'm also going to go away and read up on bisulfite sequencing in plants (I obviously really need too).
Yes I meant detecting the CpG islands. Looking at my sequence I think I agree with Methprimer (that there is a CpG Island at the end of my promoter and the beginning of my enchancer) because it is very CpG rich.
Thank you so much for your help and very prompt responses. You've been very helpful.
It is good to know there is a program for designing plant bisulfite PCR primers.
Another thought is since every cytosine could be methylated and sometimes there is no C-free region for picking primers, in this case you can design degenerate primers to cover both methylated and unmethylated alleles.
Yes I'm really pleased I found it but at the same time annoyed at myself for not searching for it sooner.
It looks like the program designs the primers so that they have a degenerate base at each position with a cystosine (either Y or R depending upon their direction). I'm much more hopeful that they'll work this time. I'll give them a try once I recieve them and let you know if they've worked.
Thanks again for all your help.
Just to let you guys know that I tried the Kismeth primers and (apart from a troublesome area of my construct) they worked really well with my high-fidelity polymerase. I haven't cloned and sequenced them yet but the bands are the right size and there's no amplification in my WT plants, so fingers crossed.
Thanks again for your help,