Question: H3K4Me3 ChIP-Seq signal at promoter
2
gravatar for Dave Tang
3.6 years ago by
Dave Tang180
Australia
Dave Tang180 wrote:

Hello,

below is a UCSC Genome Browser screen shot centred on the start of the ACTB gene that shows ENCODE ChIP-Seq signal for H3K4Me3 for the HSMM cell line (I turned off all other cell lines).


 

 

 

 

My question is, what is the cause of the dip near the promoter of this gene? The H3K4Me3 histone mark is associated with promoters and if anything, I would expect to see an enrichment at the promoter. Is this due to poised RNA polymerases binding at the TSS and thus somehow affecting the antibody binding? How does this affect peak calling?

Thanks in advance.

Dave

chip-seq h3k4me3 • 2.8k views
ADD COMMENTlink modified 2.9 years ago by abishai.dominic0 • written 3.6 years ago by Dave Tang180
1

Do you have access to the input fraction data from the same experiment ? If so, you can have a look at it and see if there is some kind of bias in that region.

Also, your screenshot doesn't show values higher than 150. It would be usefull to see all the data.

ADD REPLYlink written 3.6 years ago by Carlo Yague4.6k

Here's another screenshot where the max is set to 400

These are the ENCODE ChIP-Seq experiments, so there should be some input fraction somewhere. I'll try comparing the signals.

ADD REPLYlink written 3.6 years ago by Dave Tang180

Do other cell lines show that or just this one? If more show it then it could just be a result of GC bias.

ADD REPLYlink written 3.6 years ago by Devon Ryan91k

Here's the same locus with all the cell lines turned on:

At GAPDH

 

ADD REPLYlink written 3.6 years ago by Dave Tang180
1

My guess is that this is a GC bias. Have a look at the input as carlo.ankou suggests.

ADD REPLYlink written 3.6 years ago by Devon Ryan91k
2
gravatar for Chirag Nepal
3.6 years ago by
Chirag Nepal2.2k
Copenhagen
Chirag Nepal2.2k wrote:

I think it is showing the nucleosome free region, where signals at two sides represent divergent transcription.

ADD COMMENTlink written 3.6 years ago by Chirag Nepal2.2k

So it's the lack of nucleosomes due to transcription and therefore lack of histones and hence no signal?

ADD REPLYlink written 3.6 years ago by Dave Tang180

Yes, i think so.

ADD REPLYlink written 3.6 years ago by Chirag Nepal2.2k

I think Chirag Nepal is right. You'll most likely find a big peak of Pol II right where the H3K4me3 signal dips.

 

ADD REPLYlink modified 3.6 years ago • written 3.6 years ago by jotan1.2k

Why is that there is two peaks in the upstream and the downstream from the TSS ?

ADD REPLYlink written 2.9 years ago by abishai.dominic0
0
gravatar for abishai.dominic
2.9 years ago by
abishai.dominic0 wrote:

The article - http://www.nature.com.ezproxy.library.tamu.edu/nature/journal/v419/n6905/pdf/nature01080.pdf explains that active genes are marked by H3K4me3 which is opposite of what you are explaining here. Can you please elaborate on your explanation?

ADD COMMENTlink written 2.9 years ago by abishai.dominic0
1

H3K4me3 favours histone acetylation which in turn promotes histone depletion. So the more H3K4me3 there is, the less H3 and the less H3K4me3 there is. This makes sense because chromatin remodelling is a highly dynamic process.

In ChIP-qPCR, one solution is to look at the H3K4me3/H3 ratio from the same chromatin.Usually you will see H3 decreases at gene promoters and the ratio H3K4me3/H3 increases (even if raw H3K4me3 decreases too).

If you want more information, I suggest to take a look at the pioneer work of Buratoswki, for instance, Kim & Buratowski 2009.

ADD REPLYlink written 2.9 years ago by Carlo Yague4.6k

So are you suggesting that the dip in the peak at the TSS (between the downstream and the upstream regions) actually signify higher H3K4me3?

If that is the case, how there is that there is higher levels of H3K4me3 at the upstream and downstream regions from the TSS ?

Thank you.

ADD REPLYlink written 2.8 years ago by abishai.dominic0
1

So are you suggesting that the dip in the peak at the TSS (between the downstream and the upstream regions) actually signify higher H3K4me3?

No. What I'm saying is that even if there is less raw H3K4me3 at the TSS, it is likely that there is also less H3. And it is possible that the remaining H3 are more trimethylated. You need to look at the H3 level over that region to get an idea.

For instance look at the folowing table. What you see in your genome browser is the raw H3K4me3 (4 time less at the TSS than at the gene body), but what matters is the ratio : in this example 50% of all H3 are trimethylated at TSS but only 10% are trimethylated at the gene body

             TSS     Gene
H3K4me3       5       20
H3           10       200
ratio       0.5       0.1
ADD REPLYlink modified 2.8 years ago • written 2.8 years ago by Carlo Yague4.6k

Dear Carlo,

Thank you very much for helping me understand ! I really find this very helpful, Finally I understand what it means. I think the articles should show the ratio of methylated/unmethylated histones from now on just than showing the peaks.

But there is still one question. The article http://www.ncbi.nlm.nih.gov/pubmed/21847099 explains that

1) H3K4me3 is associated with active enhancer regions. But other articles like http://www.ncbi.nlm.nih.gov/pubmed/26317464 summarize that H3K4me3 is low/devoid in enhancers but high only in promoter regions. Is this chromatin signature still up for debate or which signature is widely accepted?

2) The article also explains that presence H3K4me3 increases POLII accumulation. If the raw H3K4me3 is reduced at the TSS compared to the other gene regions due to low histones, how does POLII bind to the TSS ?

Thank you very for explaining.

ADD REPLYlink written 2.8 years ago by abishai.dominic0

Glad I could help. :)

1) Sorry but I don't know much about enhancers. Can't really answer this one.

2) For me, things are more complicated than just "H3K4me3 increases POLII accumulation" : First H3K4 methylation depends on Pol II transcription (via the interaction of Set1 with Pol II Rpb1 CTD S5P). Secondly, H3K4 methylation by Set1 is both an activator and repressor of transcription, with H3K4me2 more of a repressor through the recruitment of HDAC (histone deacetylases) and H3K4me3 more of an activator trough the recruitment of HAT (histone acetyl-transferase). In fine, histone acetylation leads to histone depletion so the PIC (Pol II initiation complex) can bind the DNA and there is transcription.

how does POLII bind to the TSS ?

In short, Pol II doesn't need H3K4me3 to bind the TSS. What Pol II needs is a region depleted in nucleosome/histones and H3K4me3 helps with that.

This is complicated stuff, I agree.

ADD REPLYlink modified 2.8 years ago • written 2.8 years ago by Carlo Yague4.6k

Because there's less H3 to begin with. The whole region is enriched for H3K4Me3, you just see a divot at the TSS because the H3 needs to make way for PolII binding. The phrase "the TSS is enriched for H3K4me3" doesn't mean that you have to see a focal peak exactly at the TSS. This is a histone modification, it's a broader noisier thing.

ADD REPLYlink written 2.8 years ago by Devon Ryan91k

Can you add any references which can go with your explanation please?

ADD REPLYlink written 2.8 years ago by abishai.dominic0

Feel free to search pubmed or talk to anyone else in the field.

ADD REPLYlink written 2.8 years ago by Devon Ryan91k
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