Imprinted gene AND DMR - (Apr/20/2006 )
Hi,I am a new here and I have been confused with such questions for a long time.
I will work on some special imprinted gene(eg.H19,IGF2) to detect their Mathyl-status in DMRs with BSP.But I am not sure with the DMR(differentially methylated regions),what does this mean?
How can I find the DMRs in a gene? What's the difference between the DMRs and the CpGs ?
Thanks
BlackBlue
DMR stands for differentially methylated region and this means that there is parental specific methylation at that locus.
So take a hypothetical locus that is a DMR, in the normal case the paternal allele is methylated while the maternal allele is unmethylated and in BSP you will see approx 50% of your clones methylated and 50% unmethylated. deviations from this may suggest abberant methylation. If it is a DMR and you get 100% methylated or 100% non-methylation again, this is abberent and could well be lethal.
In the case of H19 Igf2, I always get confused which is methylated or not. need to go find a review article!
Nick
Thank you for clear my mind.
I konw the imprinted gene is mono-allelic expression ,the different mathyl-status between parental genes is so called DMR,right?
Another questions is how can I find methylated locus?eg,Igf2 and Peg3 are paternally expressed genes and Igf2R ,Gnas maternally expressed.Shall I analysis the total gDNA to find whole CpG islands distribution or only , just the promotor region for BSP?
So take a hypothetical locus that is a DMR, in the normal case the paternal allele is methylated while the maternal allele is unmethylated and in BSP you will see approx 50% of your clones methylated and 50% unmethylated. deviations from this may suggest abberant methylation. If it is a DMR and you get 100% methylated or 100% non-methylation again, this is abberent and could well be lethal.
In the case of H19 Igf2, I always get confused which is methylated or not. need to go find a review article!
Nick
There is no easy answer to this. The best way is to consult the literature for the candidate genes you are interested in. Some imprinted genes have been very well studied (eg. H19 and IGF2) and there is plenty of information on how their regulation is controlled. Others are less well studied and it is more difficult to get good information. Imprinted genes can also be isoform and/or tissue specific in their expression (eg. MEST, GRB10) and this complicates things further. I'd suggest investing a good deal of time reading about the imprinted genes you're interested in (and be careful to distinguish between what is going on in mice and humans as it is not always the same - eg. igf2r imprinting occurs in mice but not humans).
The paternal DMR is methylated, the maternal one unmethylated....
There is no easy answer to this. The best way is to consult the literature for the candidate genes you are interested in. Some imprinted genes have been very well studied (eg. H19 and IGF2) and there is plenty of information on how their regulation is controlled. Others are less well studied and it is more difficult to get good information. Imprinted genes can also be isoform and/or tissue specific in their expression (eg. MEST, GRB10) and this complicates things further. I'd suggest investing a good deal of time reading about the imprinted genes you're interested in (and be careful to distinguish between what is going on in mice and humans as it is not always the same - eg. igf2r imprinting occurs in mice but not humans).
Yes ,thanks for your suggesions.My boss is abroad and I totally have no idea how to go no.
As you've pointed ,I am not willing to check a new imprinted gene.The latest information is listed this link:http://igc.otago.ac.nz/Search.html.No more than 50 imprinted gene in human and over 100 in mose are founded
I just want to konw if I study H19 ,which sequence shall I pick out for BSP? in other words,generally say :H19 has been methylated ,How can I prove it ?the H19 gene may contains several CpG- enriched regions,How can I say this gene has been methylated? From the promotor region or other.....
To further clear my question,I put an attachments below.....
If I recall correctly the H19 DMR in humans extends from the promoter to about 6kb upstream. The CTCF (insulator) binding sites that influence the expression of H19 and IGF2 lie within this region. If these binding sites are methylated (usually on the paternally inherited c'some) then the methylation extends along the DMR into the H19 promoter and H19 is silenced. You could therefore study the promoter region as you have suggested.
The attachment included in your previous post shows bisulphite sequencing data in the mouse. It shows that between 11.5 and 12.5 days post conception the igf2 and snrpn DMRs have become demethylated in the primordial germ cells (demonstrating that the imprint has been reset in these cells). The somatic cells show differential methylation at 12.5 days indicating that demethylation is specific to the primordial germ cells.
Hope this helps.
Dear karyotyper
I think I gradually understand now.
The post cited is from paper on PGC reprograming study.As you pointed,the H19 DMR lies from the promoter to about 6kb upstream.Please tell me how that table is made? I know the oval means the CpG island, the filled has been methylated and the blank not. which confused me is if I get the sequencing results,how can I transform the datas into that table? By some soft or else?And I can't understand what the parallel line(in the attachment) means(all the CpG lie in same locus)?why not straight line to show the DMR?
Hope your reply soon,THANKS!
The attachment included in your previous post shows bisulphite sequencing data in the mouse. It shows that between 11.5 and 12.5 days post conception the igf2 and snrpn DMRs have become demethylated in the primordial germ cells (demonstrating that the imprint has been reset in these cells). The somatic cells show differential methylation at 12.5 days indicating that demethylation is specific to the primordial germ cells.
Hope this helps.
if all you want to find out is the methylation status of H19, BSP may not be necessary and traditional HpaII/MspI restriction digest and southern blotting can be employed as this would be easier to perform that BSP if you have not done either before. There are many refs that use this technique to demonstrate methylation of H19. Pubmed is your friend.
Nick
BlueBlack
Each oval represents a CpG. Each line of ovals represent the CpGs that were sequenced within that clone. There is a series of parallel lines because each line represents a separate sequenced clone (for the same region). It is common to sequence 8 or 10 clones for a particular region to study the methylation profile following BSP. When studying imprinted genes it is common to sequence even more so a representative sample of maternal and paternal chromosomes are examined.
I'm no technical expert in this area and am only just starting off myself (verrrry sloooowly!!!). I do, however, have access to great people with experience which certainly helps. It is probably easier to approach this problem in simplier ways as Methylnick has suggested - i suppose it depends on what exactly you're trying to do. Keep reading is all I can suggest as there is heaps of published stuff on H19.
Also see this website for software for bisulphite sequencing BiQ Analyzer There are others available as well.
Good luck again.
Oh....Thank you for your return.
I've learnt a lot from you TWO. I am a new in this field and nobody else study imprinted gene in my lab.I think heteroduplex may be the cause of the heterology of BSP results ( parallel lines) right?
I will keep reading Refs related.Thank you again.
BlueBlck
BlueBlack
Each oval represents a CpG. Each line of ovals represent the CpGs that were sequenced within that clone. There is a series of parallel lines because each line represents a separate sequenced clone (for the same region). It is common to sequence 8 or 10 clones for a particular region to study the methylation profile following BSP. When studying imprinted genes it is common to sequence even more so a representative sample of maternal and paternal chromosomes are examined.
I'm no technical expert in this area and am only just starting off myself (verrrry sloooowly!!!). I do, however, have access to great people with experience which certainly helps. It is probably easier to approach this problem in simplier ways as Methylnick has suggested - i suppose it depends on what exactly you're trying to do. Keep reading is all I can suggest as there is heaps of published stuff on H19.
Also see this website for software for bisulphite sequencing BiQ Analyzer There are others available as well.
Good luck again.