Question: what is the motivation behind Pol2 chip-seq data?
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gravatar for Affan
5.0 years ago by
Affan290
Canada
Affan290 wrote:

I am reading a few papers on histone modifications where the data comes from chip-seq experiments. But  most if not all of the papers also get data on RNA polymerase 2.

I see why we chip-seq histone modifications. It gives us the positions of these modifications within the genome and based on gene annotation, we can see if these modifications exist near expressed or inhibited genes.

However, RNA polymerase 2 is just a protein that transcribes genes. Why do we care about where it is (ie, where it is bound) at the moment chip-seq is run?

 

chip-seq sequence • 5.6k views
ADD COMMENTlink modified 5.0 years ago by mikhail.shugay3.4k • written 5.0 years ago by Affan290

Could it be that RNA Polymerase binds to TSS and knowing the TSS can be helpful? Genuine loud thinking because I'm bad at biology.

ADD REPLYlink written 5.0 years ago by RamRS25k
2
gravatar for bede.portz
5.0 years ago by
bede.portz490
United States
bede.portz490 wrote:

You write: However, RNA polymerase 2 is just a protein that transcribes genes. Why do we care about where it is (ie, where it is bound) at the moment chip-seq is run?

The very point of histone modifications, transcription factor binding, the binding of pausing, elongation, and termination factors, etc is to regulate gene expression, and Pol II is the very machine responsible for protein coding gene expression in eukaryotes. Those studies linking the role of some histone modification, nucleosome position, factor binding, chromatin state, etc. to transcription would be better served including an analysis of the location of Pol II, and depending on aspects of the way the experiment was conducted, such knowledge may actually be better than an expression array or RNA-seq data. 

Further, Pol II is not, "just a protein that transcribes genes," but rather it is arguably THE regulatory nexus for development/stimulus-response networks/homeostatis or whatever. Think about it; pol II is at once the terminal receiver of any signalling event regulating protein coding gene expression AND the initial step in the response to said event. It is very literally the inflection point between stimulus and response! This protein is itself highly modified in order to coordinately regulate every step of the transcription cycle (initiation, pausing, elongation, termination, recycling of the polymerase for subsequent transcription) via myriad post-translational modifications that we have only begun to understand. Pol II recruits factors to itself involved with all the aforementioned steps as well as RNA processing and histone modication. It is far more the supervisor of a factory  than it is "just a protein that transcribes genes." 

In higher eukaryotes Pol II is regulated post initiation, via promoter proximal pausing, and the position of Pol II at promoters relative to gene bodies is revealing in ways that are difficult if not impossible to appreciate using data derived from RNA-seq/microarray or nascent transcript sequencing which fail to accurately juxtapose promoter vs gene body Pol II levels in ways that reflect biology. 

 

ADD COMMENTlink modified 5.0 years ago • written 5.0 years ago by bede.portz490
1
gravatar for Saulius Lukauskas
5.0 years ago by
London, UK
Saulius Lukauskas530 wrote:

While post-translational histone modifications can serve as a general rule of thumb to mark active/repressed genes, the exact mechanisms of how they work are not that well understood in general. For instance, some repressive marks can co-occur with active ones at certain loci. 

RNA Pol-II binding, however, is a very clear cut sign of gene transcription occurring. If RNA Pol-II was not recruited to locus, then no transcription would occur at all, regardless of histone modifications (or other things) in the locus. This is the reason, I assume, the papers are ChIP-ing the Pol-II as well -- to show that transcription is happening directly. It is hard to tell without you being more specific which papers are these.

By the way note that recruitment of Pol-II to a locus is not sufficient to transcribe a gene. Namely, Ser5 needs to be phosphorylated to initiate transcription and then further Ser2 phosphorylation is needed for transcription elongation to occur. Keep that in mind, as some papers ChIP the specific phosphorylation variants of RNA Pol-II as well.

 

ADD COMMENTlink modified 5.0 years ago • written 5.0 years ago by Saulius Lukauskas530
0
gravatar for dariober
5.0 years ago by
dariober11k
WCIP | Glasgow | UK
dariober11k wrote:

On reason for ChIP'ing RNA Pol II might be to know where the polymerase stalls, for example as a consequence of chromatin structure or other proteins or factors that cause Pol II to pause. Having said that, if a paper is nicely written the authors should say why they have looked at RNA Pol II.

ADD COMMENTlink written 5.0 years ago by dariober11k
0
gravatar for mikhail.shugay
5.0 years ago by
mikhail.shugay3.4k
Czech Republic, Brno, CEITEC
mikhail.shugay3.4k wrote:

When dealing with ChIP-Seq peaks for your transcription factor of interest, in order to reduce the number of false-positives it is usually wise to consider a) the chromatin is open -> DNAse hypersensitivity track and b) the polymerase is actually binding to the promoter -> Pol2 binding track. You can also verify alternative promoters with this track.

ADD COMMENTlink modified 5.0 years ago • written 5.0 years ago by mikhail.shugay3.4k

What is the importance of DNase hypersensitivity track? And what do you mean by false-positives? Sorry I am a math major and just dived into bioinformatics for my MSc research.

ADD REPLYlink written 5.0 years ago by Affan290

DNase hypersensitivity areas could be accessed by transcription factors (http://en.wikipedia.org/wiki/DNase_I_hypersensitive_site ). Given that all of this signals are necessary for a gene activation by a transcription factor, the intersection of them increases the reliability of the association between a given transcription factor binding and gene up-regulation.

ADD REPLYlink modified 5.0 years ago • written 5.0 years ago by mikhail.shugay3.4k
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