7 replies to this topic

[ChIPgal6]

Thank you for this fantastic forum! I needed help with interpreting my contradictory ChIP results. My treatment is up regulating my gene of interest as seen by mRNA levels in Jurkat cells. When we do ChIP, signals from ChIP-qPCR are showing enrichment for acetylation at lysine 9 and 14 (H3Ac), trimethylation at lysine 4 (H3K4Me3). However, histone H3 is showing enrichment as well. What do you think is going on? Why is there enrichment for total H3?

[KPDE]

ChIPgal6, on 20 September 2010 - 01:41 PM, said:

Thank you for this fantastic forum! I needed help with interpreting my contradictory ChIP results. My treatment is up regulating my gene of interest as seen by mRNA levels in Jurkat cells. When we do ChIP, signals from ChIP-qPCR are showing enrichment for acetylation at lysine 9 and 14 (H3Ac), trimethylation at lysine 4 (H3K4Me3). However, histone H3 is showing enrichment as well. What do you think is going on? Why is there enrichment for total H3?

When you say you see enrichment for total H3, do you mean you see an increase with the treatment?

[ChIPgal6]

KPDE, on 20 September 2010 - 02:42 PM, said:

ChIPgal6, on 20 September 2010 - 01:41 PM, said:

Thank you for this fantastic forum! I needed help with interpreting my contradictory ChIP results. My treatment is up regulating my gene of interest as seen by mRNA levels in Jurkat cells. When we do ChIP, signals from ChIP-qPCR are showing enrichment for acetylation at lysine 9 and 14 (H3Ac), trimethylation at lysine 4 (H3K4Me3). However, histone H3 is showing enrichment as well. What do you think is going on? Why is there enrichment for total H3?

When you say you see enrichment for total H3, do you mean you see an increase with the treatment?

Yes, I see an increase in the total H3 signal with the treatment.

[Mighty Mouse]

ChIPgal6, on 20 September 2010 - 01:41 PM, said:

Thank you for this fantastic forum! I needed help with interpreting my contradictory ChIP results. My treatment is up regulating my gene of interest as seen by mRNA levels in Jurkat cells. When we do ChIP, signals from ChIP-qPCR are showing enrichment for acetylation at lysine 9 and 14 (H3Ac), trimethylation at lysine 4 (H3K4Me3). However, histone H3 is showing enrichment as well. What do you think is going on? Why is there enrichment for total H3?

Here's just a wild stab in the dark...maybe your treatment is increasing the likelihood of your region of interest interacting with an enhancer region? Perhaps what you are picking up with your ChIP (assuming you are doing FA crosslinking and not native ChIP) is additional histones from an enhancer region via tertiary or quaternary binding? Probably unlikely, but it's the first thing that comes to mind.

MM

[ChIPgal6]

Here's just a wild stab in the dark...maybe your treatment is increasing the likelihood of your region of interest interacting with an enhancer region? Perhaps what you are picking up with your ChIP (assuming you are doing FA crosslinking and not native ChIP) is additional histones from an enhancer region via tertiary or quaternary binding? Probably unlikely, but it's the first thing that comes to mind.

MM
[/quote]

Thank you Mighty Mouse! What is FA crosslinking vs. native ChIP? I crosslink proteins to DNA with 10 minute formaldehyde treatment followed by 5min quenching with glycine. I chop up my DNA with micrococcal nuclease into 200-400bp fragments, which I use to set up IP with. Pulled down DNA is washed, reverse-crosslinked and purified before PCR.

[KPDE]

ChIPgal6, on 02 October 2010 - 02:41 PM, said:

Thank you Mighty Mouse! What is FA crosslinking vs. native ChIP? I crosslink proteins to DNA with 10 minute formaldehyde treatment followed by 5min quenching with glycine. I chop up my DNA with micrococcal nuclease into 200-400bp fragments, which I use to set up IP with. Pulled down DNA is washed, reverse-crosslinked and purified before PCR.

FA crosslinking is what you've used in your ChIP so far (FA = formaldehyde).  Native ChIP is ChIP without crosslinking, which is possible because of how tightly the nucleosomes bind to the DNA.

Where are you seeing the increases in H3 and the two acetylations?  Transcription start site, promoter, body of the gene ...?

Edited by KPDE, 02 October 2010 - 06:27 PM.

[ChIPgal6]

KPDE, on 02 October 2010 - 06:26 PM, said:

ChIPgal6, on 02 October 2010 - 02:41 PM, said:

FA crosslinking is what you've used in your ChIP so far (FA = formaldehyde).  Native ChIP is ChIP without crosslinking, which is possible because of how tightly the nucleosomes bind to the DNA.

Where are you seeing the increases in H3 and the two acetylations?  Transcription start site, promoter, body of the gene ...?

The increase in H3 and H3 acetylations is seen in the 185bp promoter region adjacent to the TSS as well as the TSS.

[angelawu]

In the past I have seen many people normalize their histone IP to the total H3 IP. Since the total H3 should stay the same if you use the same number of cells, it is possible that the increase in total H3 that you see from no treatment to treatment is simply from the one ChIP experiment working better than the other one.  In this case, if you normalize all your histone IPs to the total H3 amount, you can see if you get increase upon treatment using this normalized value.

ChIPgal6, on 21 October 2010 - 12:46 PM, said:

KPDE, on 02 October 2010 - 06:26 PM, said:

ChIPgal6, on 02 October 2010 - 02:41 PM, said:

FA crosslinking is what you've used in your ChIP so far (FA = formaldehyde).  Native ChIP is ChIP without crosslinking, which is possible because of how tightly the nucleosomes bind to the DNA.

Where are you seeing the increases in H3 and the two acetylations?  Transcription start site, promoter, body of the gene ...?

The increase in H3 and H3 acetylations is seen in the 185bp promoter region adjacent to the TSS as well as the TSS.