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Old 08-18-2011, 02:35 PM   #1
Wonko
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Location: Seattle

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Default ChIP-Seq with HA-tagged protein

Hi everybody,

I am trying to define site-specific and off-site binding of a DNA binding protein in human cells (at this point HEK-293 and K-562 cells).

The protein of interest is not expressed naturally in human cells, i.e. it has to be expressed heterologously. Furthermore, at this point there is no antibody available for the protein. Therefore I am using a N-terminal HA tag for ChIP with a ChIP-grade antibody.

Here's a quick run down of my protocol:

- transfect (HEK-293) or transduce (K-562) cell with expression vector
- growth for 48h
- fix cells 5x10exp7 with formaldehyde (1% for 10min) quench with Gly
- QC: WB with the a-HA antibody that I use as well for ChIPing. Protein expression can be verified under a fluorescent light microscope or by FACS since my protein is linked via a T2A sequence to mCherry.
- cell lysis and sonication on a Covaris AFA instrument
- binding of the HA-tagged protein to the a-HA antibody immobilized on magnetic beads (protein G) ON at 4°C.
- wash, crosslink reversal and elution
- ProteinaseK and RNase treatment
- QC: size distribution of the sheared DNA
- DNA end repair, addition of A bases, ligation of (barcoded) sequencing adapters and PCR amplification
- QC: amplification is done on a light-cycler using SYBR-green to monitor the emergence of amplification products.
- PAGE purification of the amplified libraries.
- QC of the libraries on an Agilent Bioanalyzer; I aim for fragments of 200-400bp.
- 76bp SE Illumina sequencing

I get 20-60 mil reads that I map back to GRCh37/hg19 using bowtie or bwa. Peak calling is done with MACS.

My problem is, that I get a LOT of background. One reason for this is already the low percentage of cells that initially express my protein. Based on mCherry expression I can say that it's only about 1/3 of the total number of cells that light up under the microscope. In order to alleviate this problem I've put together a new expression plasmid that has a selection marker and is Tet inducible so that I can hopefully increase this number. I didn't get to try it out yet though. Cell sorting is not an option because of the large number of cells.
On that note, does anybody have a rough idea of the number of molecules/cell of a protein one needs for a reliable ChIP-Seq experiment?

Besides the amount of protein I suspect that the main problem is the a-HA antibody that I am using for the ChIP. Has anybody already used the HA-tag for ChIP?
Which tags might be better choices in this setting? I am considering to switch to another tag, namely GST or His. I've seen a study in S. cerevisiae that featured a myc-specific antibody for ChIP but I guess if I use that in human cells I will pull down all the c-myc targets.

It is not known if and where my protein of interest binds in the human genome - that's exactly what I am trying to figure out. As long as I can say with confidence that there are no binding sites I'd be happy with that. As positive control I use a known binding sequence for my protein that I inserted into the HEK-293 genome (I don't have a positive control site in K-562 cells).
I do not see this positive control after peak calling. At this point I fear that potential peaks might either not be visible because of insufficient enrichment (either caused by the low number of protein molecules per cell or inefficient pull-down of bound sequences) or because they are covered by the strong background.

I'd appreciate any suggestions,

Wonko

Last edited by Wonko; 08-18-2011 at 02:39 PM.
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Old 06-25-2012, 02:04 AM   #2
Benji Price
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Default HA (or other)-tag ChIP

Hi Wonko,

I am preparing an experiment very similar to the one that you described here sometime ago: ectopic expression of a HA-tagged and FLAG-tagged protein not present in the host cellular background (293, Hela), induced by TET-ON expression system. I hope everything went fine for you at the end.

I wondered if you finally solved by yourself the questions that you rised in your post, mainly that about the suitability of HA-tag for ChIP-seq assays. I would greatly appreciate any further information you could consider relevant for avoiding any pitfall you encountered in the road to obtaining your desired results.

Besides, I had a previous model tagged with myc (originally developed for other purposes) in which I have done some ChIP-qPCR pilot experiments. I have found that, despite that 90% of the ectopic induced protein being inside the nucleous after only 4h of doxycycline addition (as proved by Immunofluorescence), I am not able to detect enrichment of promoters used as positive controls. Instead, I am detecting a reduction of the myc-ChIPed DNA in the Doxy-induced conditions (0.5 µg/ml Doxycycline), which is leading me to think that a vast portion of the ectopically expressed protein might be not bound to the DNA (probably because of saturation of the binding sites) and could be competing with the DNA-bound fraction during de Chromatin-Dynabeads-Antibody incubation (5-6 µg DNA + 25 µl dynabeads + 5 µg antibody). So, I am thinking that titrating the Doxycycline concentration until obtaining a “physiological-like” protein level could be crucial for this kind of assays.

Thank you in advance. Any ideas from other members would be also appreciated.

Benji.
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Old 06-25-2012, 12:25 PM   #3
Wonko
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Default

Hi Benji,

I spent quite some time trying to make the ChIP work with the HA tag but in the end I decided to cut my losses and I switched to another tag. Either it was the antibody that I was using or the unsuitability inherent to the HA tag itself, I could never get any convincing data.

After looking around a bit more I stumbled over a paper in BMC Molecular Biology (Kolodziej KT (2009) BMC Molecular Bilogy, http://www.ncbi.nlm.nih.gov/pubmed/19196479) describing the successful utilization of a tandem tag consisting of a V5 tag and a BirA biotinylation target sequence for ChIP. Unlike for the HA tag, there are even beads commercially available from Sigma Aldrich with antibody against the V5 tag. Using the V5 beads I get ca. tenfold enrichment of a putative genomic target site using the Ct method (Litt MD (2001) The EMBO Journal, http://www.ncbi.nlm.nih.gov/pubmed/11331588).

In addition to changing the antibody I modified my protocol a bit in order to reduce background/increase signal:
- I now use a Tet inducible expression construct (just like you :-))
- chromatin is pre-cleared before the ChIP-step
- ChIP samples are subjected to an exonuclease treatment (ChIP-exo, Rhee HS (2011) Cell, http://www.ncbi.nlm.nih.gov/pubmed/22153082)

As an alternative to the V5/Bio tag I put as well together an expression construct with the HaloTag tethered to my protein (http://www.promega.com/resources/pro...stem-protocol/). It looks promising according to the description but I did not get to use it yet.

Thank you very much for your insights into your experiments with the myc-tag. I just used that tag for Western blotting and never tried to do a ChIP experiment with it. So it was very interesting to hear about your experience and your thoughts about the result.

I hope my answer is somewhat helpful. Best,

Wonko

Last edited by Wonko; 06-25-2012 at 12:51 PM.
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Old 06-25-2012, 12:50 PM   #4
Benji Price
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Default

Thank you Wonko, it is really useful information. I will go on with the HA-inducible expression, as I have everything ready and it wont take long to develop the clones. However, I will follow your advice in case of not obtaining good results.
Anyway, and although my experience with ChIP is limited, I still think that the problem could be the artefactual protein levels achieved by ectopic overexpression rather than the tag itself. So I will also try to reduce doxy levels.
Thank you again. Regards,
Benji.
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Old 06-24-2014, 08:45 AM   #5
icastro
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Hello Benji,
Wondering what was your experience with the HA tag and ChIP-Seq compatibility. I am too looking for a Tag for my protein of interest and now this can be anything (V5, GFP, HA), so would very much appreciate your thoughts on this and avaibility of good ChIP-Seq anti-tag antibodies.
Thank you,
Ines
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