Finding out what sequence to study by ChIP - (Jul/28/2008 )
Well, as we are hybridising our ChIP DNA to an array we want to minimise non-specific binding. For example: say if our histone binds to a region which is 300bp, and yet our DNA fragments are 2000bp long, that gives an extra 1700bp of DNA that could hybridise to our array but isn't actually bound to the histone.
Does that make sense?
I have been drinking wine whilst packing in preparation for moving house. Ahhh bioforum gives me a nice break 
Clare
Thanks Clare,
Speaking of what region to study, before when I was studying DNA methylation, I picked CpG islands to study, does that apply to histone trimethylation as well? Or there is usually another chromatin mark where people can predict histone binding (and thus the region to design primers for)??
Thanks Clare,
Speaking of what region to study, before when I was studying DNA methylation, I picked CpG islands to study, does that apply to histone trimethylation as well? Or there is usually another chromatin mark where people can predict histone binding (and thus the region to design primers for)??
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Hi there,
Sorry for the late reply - I just moved house and only got the net connected today (YAY! Finally!).
I am not expert in trimethylation. We are just looking at regions about 200kb upstream and 100kd downstream of the genes we are interested in to see what comes up. So far, with H3K27me3 regions are upstream/downstream...unlike our H3K9 which is more near start sites and within 1st introns.
Clare
Speaking of what region to study, before when I was studying DNA methylation, I picked CpG islands to study, does that apply to histone trimethylation as well? Or there is usually another chromatin mark where people can predict histone binding (and thus the region to design primers for)??
Hi there,
Sorry for the late reply - I just moved house and only got the net connected today (YAY! Finally!).
I am not expert in trimethylation. We are just looking at regions about 200kb upstream and 100kd downstream of the genes we are interested in to see what comes up. So far, with H3K27me3 regions are upstream/downstream...unlike our H3K9 which is more near start sites and within 1st introns.
Clare
200kb upstream and 100kb downstream??
that means the H3K27me3 region are very far from the gene??
I want to clarify one thing....
200kb upstream means from -200kb position until start site are all trimethylated? or just at -200kb position has a single small region being trimethylated?
If it's the latter case, how could I possibly design primers to do PCR? It sounds like so random
Sorry for the late reply again! This time I have been on holiday wOOt!
We look at the region -200kb to +100kb via chip-chip. Specific areas come up as being trimethylated and I zoom in on those areas to design primers for qPCR validation.
Do you have any idea where your gene is trimethylated??
Clare
[/quote]
200kb upstream and 100kb downstream??
that means the H3K27me3 region are very far from the gene??
I want to clarify one thing....
200kb upstream means from -200kb position until start site are all trimethylated? or just at -200kb position has a single small region being trimethylated?
If it's the latter case, how could I possibly design primers to do PCR? It sounds like so random
[/quote]
wOOt!We look at the region -200kb to +100kb via chip-chip. Specific areas come up as being trimethylated and I zoom in on those areas to design primers for qPCR validation.
Do you have any idea where your gene is trimethylated??
Clare
No idea at all where the trimethylation might be, and I dun think we have a very good budget to do chip-chip
that's the problem in research, you dun always have the money....
so I am looking for a cheaper (yet valid) method to find out the histone trimethylation (probably realtime PCR) at different position
if the range is -200kb to +100kb, it's simply not feasible
What should I do?
What should I do?
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Is there anything published about your gene and trimethylation? Anything that can give you a little clue?
You can also read general trimethylation papers and see if a pattern emerges and perhaps guess where it's happening?
Is there anything published about your gene and trimethylation? Anything that can give you a little clue?
You can also read general trimethylation papers and see if a pattern emerges and perhaps guess where it's happening?
apparently not
there's not much information about most of them
and especially if I am studying 10 genes, it's unlikely that it's been all studied before.
Is there anything published about your gene and trimethylation? Anything that can give you a little clue?
You can also read general trimethylation papers and see if a pattern emerges and perhaps guess where it's happening?
apparently not
there's not much information about most of them
and especially if I am studying 10 genes, it's unlikely that it's been all studied before.
And that's also why I asked before whether I should sonicate the DNA into larger fragments and design primers that covers a broader area
I just read something:
2 papers talked about position of histone methylation
Wiencke JK et al, Oncogene 27, p2412
Liang G et al, PNAS 101, p7357
H3K9Ac, H3K4me2, H3K9me3 are mostly within 1kb of the transcriptional start site (TSS) of genes in human cells
Why they studied H3K4me2 instead of H3K4me3???
I thought the H3K4me3 is the activation signal, based on Mikkelsen TS et al, Nature 448, p553, but not H3K4me2
I don't know much about H3K4me2
I think maybe it's reasonable to look around -5kb to +1kb
but in terms of what histone modification to look at.....
If my hypothesis is histone modifications affect expression of my target genes,
H3K27me3 + H3K9me3 + H3K4me3 maybe??
what about H3K4me2 and H3K9Ac, or H3K36, H3K79, H4K20???
Seems like too much, with my limited resources
Please give me some advice, thanks a lot!!
Hi again,
Sorry but I really don't have time to be reading up about which histone modifications you should be studying. Do you have a supervisor or colleague working in a similiar area that you could ask?
Clare