FGRS: course notebook - chapter ten

 

Chromatin Immunoprecipitation

Background Information


What’s the point?


It is quite simple actually.


We are interested in the binding locations of proteins (transcription factors) that regulate gene expression.  Chromatin immunoprecipitation (ChIP) represents a technique by which we can purify small fragments of genomic DNA that represent in vivo locations of binding by factors enacting regulation on gene targets.  This technique purifies all genomic targets enabling us to characterize not one, two or a dozen binding sites - but all binding sites of a transcription factor under a specific condition.


More information on ChIP can be found at the following locations:

  1. Wikipedia

  2. Bart’s Cookbook @ Salk

  3. Molecular Station


Overall Process Description

Picture Pages Introduction

As described above, we are specifically interested in the nature of transcription factor binding to genomic loci (as depicted below):




We are not interested in just single sites of binding, however, we are interested in characterizing whole-genome binding for each transcription factor of interest.






The experimental methodology we employ is known as ChIP-seq.  This is a cutting-edge research technique that combines chromatin immunoprecipitation (ChIP) with high-throughput next-generation sequencing technologies.


The first step of ChIP-seq is the selection of a either an antibody against a protein of interest or an affinity-tagged strain.  We are utilizing six affinity-tagged (TAP) strains for each of our transcription factors of interest (ACE2, FKH1, FKH2, MBP1, MCM1, SWI4, SWI5 and SWI6).


The tag is depicted below as a small triangle endogenous to the transgenic protein.




The next step in the procedure is to capture the proteins interacting with their in vivo genomic targets.




We then utilize sonication to shear the genomic DNA.  The cross-linked proteins will remain attached to the small fragments of genomic DNA to which they were interacting.




After sonication we are able to purify the proteins of interest.  This allows us to capture our proteins of interest (along with the fragments of genomic DNA) and get rid of everything else.




Next, we reverse the cross-links in order to begin the process of purifying the DNA itself.





After a significant preparation of the DNA, the pool of purified fragments are submitted to the next-generation sequencing core facility for processing.




The sequencing facility returns millions of reads (sequenced DNA fragments) that must be mapped to specific areas of the genome through computational methods.




From these genomically mapped fragments we can establish consensus sequences that represent sites of binding.  These consensus sequences represent peaks.





Cell Growth

During this step of the process we are growing cells under controlled conditions.


In final experimentation we will be careful to grow cells to a specific OD 600 of 0.6 to 0.8 in order to ensure cells are happy, healthy and representative of normal transcriptional regulation and cell-cycle progression.  Cells experiencing environmental repression due to nutrient starvation or overcrowding are not of interest to us!


Cross-Linking of Proteins to DNA

Before we cease growth of the cells we need to do something in order to be able to study the genomic locations of transcription factor binding.  We need to lock the factors in place on the genomic DNA.  This is done by cross-linking of proteins to DNA.


Cell Lysis

With proteins cross-linked to DNA we can break the cells open releasing whole-cell extract: proteins, lipids, nucleic acids - everything.  Including: proteins bound to DNA!


Sonication

Genomic DNA bound to proteins is far too long and structured to be successfully purified by protein-IP.  Thus, we need to break the DNA up into smaller, well defined chunks.  This is done through a process of sonication.


Sonication is the act of applying sound (usually ultrasound) energy to agitate particles in a sample, for various purposes (Wikipedia).


Immunoprecipitation, Mock IP, Input DNA

The process of chromatin immunoprecipitation (ChIP) assumes that we are specifically selecting for a population of proteins that have our affinity (TAP) tag.  This specific selection is typically mediated by an antibody.  In our case, the Igg on the Igg+sepharose beads specifically binds to the Protein A that is part of the TAP tag.  By selecting for these proteins we are selecting for the fragments of genomic DNA to which they were bound.


Every experiment must have a control sample, however.


ChIP experiments have one of two options for control: Input DNA and/or Mock IP.


Your protocol directs you to do three reactions: the ChIP itself, Input DNA and Mock IP.  Thus, at least initially, in order to have a control sample for the ChIP reaction we will be doing Input DNA and Mock IP.


Input DNA will be discussed next.


If you refer to the ChIP protocol the concept is actually explained therein.  Input DNA is essentially the DNA purified by cell lysis and sonication.  It is the same DNA that is run on a gel to test the efficiency of cell lysis and sonication.


This DNA sample is set aside and processing resumes on it at the reversing of cross-links steps.  One should then ask:  What is it that Input DNA represents?


Well, just that!  Input DNA is the DNA that went through the process without any specific selection for fragments related to binding of transcription factors.


In essence, more or less, it is fractionated genomic DNA.  If we were to PCR test for regions of previously characterized binding by our transcription factor of interest would we likely produce a band?  Why, yes.  We would.  Remember, it’s genomic DNA.  All genomic loci are likely represented in the sample.


What good is it then if positive control regions will be positive?  Well, it’s all about enrichment.  Yes, this sample will have our positive control loci - but - in much lower relative quantities.  This is the essence of enrichment.  Relative to our ChIP, Input DNA will have far fewer copies.  We should be able to demonstrate a quantitative difference between our ChIP samples and our Input DNA samples (even if both are positive).


Mock IP will be discussed next.


Mock IP is a twist on the idea of Input DNA.  It asks the question “What if we processed Input DNA with beads but just not beads that are specific to our protein of interest?”.


Seems like a good idea to me.  Thus, a Mock IP is traditionally done by performing all the steps in a ChIP protocol with the exception of antibody addition.   Note that we are not using an antibody as we are relying specifically on the affinity of Igg and Protein A.  How do we perform the equivalent of a Mock IP, then?


We do this by performing two pre-clearing steps.  In essence, we are using the sepharose beads twice: once as pre-clearing and once as non-specific selection.  See the ChIP protocol for more details.


What are the relative advantages and disadvantages to Input DNA vs. Mock IP as a control for ChIP?  It’s debatable.  Some say Mock IP is a better control because it is more like the ChIP experiment and is thus a more valid control.  Critics, however, contend that the output from Mock IP is more random when compared to Input DNA and is thus an inconsistent control experiment.  Note that we are not sure whether a positive control PCR on Mock IP DNA will yield a positive result - it might and it might not.  This is the essence of the problem with Mock IP and why Input DNA is often preferred.


Washing

With our protein of interest tightly bound to beads that specifically select with it we then wash the beads (three steps, increasing stringency) in order to flush everything else away.


Elution

With all non-specific binding eliminated and all contaminants kicked out of the party we can now elute (remove) our proteins (and thus DNA fragments of interest) from the beads.


Final Precipitation Processing and Precipitation

In the end we wish to get rid of everything that is not genomic DNA fragments.  First we must reverse the cross-links between the proteins and DNA.  Next we treat with RNase A (DNase free) and Proteinase K in order to eliminate any RNA or protein contamination.  A final phenol:chloroform extraction ensures that all organic elements (including RNase A and Proteinase K) are eliminated.  The DNA is then precipitated through standard methods.


This marks the end of the experimental process but just the beginning of making sense of whether we actually purified what we intended to.



Strain Selection & Validation Experiments

In order to be sure we are actually working with the strains we believe we are working with we need to PCR (genomic DNA) and western (protein) verify each strain for inclusion of the TAP tag.


See the following research progress area (linked on the course materials page).



Growth Characterization Experiments

It is important to know whether our TAP-verified strains exhibit growth restriction as compared to wild-type (S288C).  We will use growth curves and plate-spotting to assay for growth delta.


See the following research progress area (linked on the course materials page).



Cell Lysis & Sonication Experiments

We need to be sure that cell lysis and sonication are proceeding efficiently and effectively for there to be any hope of downstream step results.  Thus, we should engage a set of experiments that allows to probe for optimization of results as focused upon these two steps. 


At the end of this process we should know which combination of cell lysis and sonication is needed to produce the fragmented DNA we can confidently take into the immunoprecipitation steps.


See the following research progress area (linked on the course materials page).



Primer Selection for ChIP-DNA Validation

You have a transcription factor with which you are working.


You expect that transcription factor to have some number of genomic binding sites.  These are places in the genome where the factor binds and regulates gene expression.  For each of the factors we are studying many sites are known (previous literature and yet more).


You have used the technique of ChIP (chromatin immunopreciptation) to purify DNA fragments that represent the actual in vivo genomic locations of binding.


Would it not be nice to perform some assay that would allow you to make heads or tails of whether your ChIP-purified DNA sample is in any way valid?


Yes, yes it would.


See the following research progress area (linked on the course materials page).



ChIP-DNA Progress

We are tracking the progress of our various ChIP prototype procedures and results.


See the following research progress area (linked on the course materials page).



ChIP-DNA Validation

We are tracking the progress of our various ChIP-DNA PCR validation experiments.  This involves the analysis of positive and negative control target loci for ChIP, Input DNA, and Mock IP samples.


See the following research progress area (linked on the course materials page).