antibody array for histone modification identification - (Sep/28/2006 )
I heard someone in a seminar talking about the use of antibody arrays in order to identify changes in histone acetylation/phosphorylation/glycosylation in response to a environnemental change. This was not really clear for me, maybe someone knows about this technique ?
As far as I understood, the idea is to spot on a microarray several antibodies specific to several modifications of the various histones. Then you "hybridize" these arrays with chromatin extracted from samples exposed to different environnemental stresses, let's say a control and a treated sample. And you expect to see differences in the level of hybridization with one or several antibodies between the two samples. Then, the next step would be to use the corresponding antibody to do ChIP on the same samples and then characterise in some way the DNA sequence which is involved and which suffer a change in histone modification in response to the environnemental stress. The idea of the guy was to use DNA micro array to identify the DNA sequences.
This techniques looks a bit complicated for me, maybe I did not get it right ??? I would think that lots of DNA sequences would immuno precipitate. Does anyone has an opinion on that ?
Thanks.
Valerie
As far as I understood, the idea is to spot on a microarray several antibodies specific to several modifications of the various histones. Then you "hybridize" these arrays with chromatin extracted from samples exposed to different environnemental stresses, let's say a control and a treated sample. And you expect to see differences in the level of hybridization with one or several antibodies between the two samples. Then, the next step would be to use the corresponding antibody to do ChIP on the same samples and then characterise in some way the DNA sequence which is involved and which suffer a change in histone modification in response to the environnemental stress. The idea of the guy was to use DNA micro array to identify the DNA sequences.
This techniques looks a bit complicated for me, maybe I did not get it right ??? I would think that lots of DNA sequences would immuno precipitate. Does anyone has an opinion on that ?
Thanks.
Valerie
That's a pretty interesting approach. I think it would be especially interesting for looking at changes during development or during early ES cell differentiation. I know of people that have done a much lower throughput way of doing this (western blots for histone mods) followed by ChIP-on-chip or QPCR profiling, but starting with an antibody array is a much better way to target global changes. Thanks for mentioning it.
valerie,
that is pretty much the gist of it, you are right.
Histone modifications and DNA methylation for that matter are dynamic modifications that could change rapidly through development and maybe even the cell cycle. I think many papers are addressing the issue of histone modification as an all or nothing approach, gene is silenced, histones at promoter reflect this, with the nature of ChIP and the techniques used I think it is still a rather rough tool that will tell you with the cells you have, the majority of them have THAT particular promoter silenced, there would be a small population that would be otherwise,
I have heard of antibody microarrays and I think this is a nice way of trying to address the issue that modifications are indeed dynamic and I would say the pool of histone modifications would change in response to stimuli and this could be detected by such an array, indeed you are right in saying many sequences will be "pulled down" and if you are fortunate enough to be in a lab that can afford high resolution tiling arrays, you could certainly start addressing what sequences are associated with the modifications that change across time and then confirm by ChIP-PCR.
Certainly a very nice way to go about a complex problem.
N