New & Noteworthy

Cutting Down on the ChIPs

January 16, 2014

We all know that potato chips are delicious.  But we also know that eating too many of them isn’t very good for our arteries or our waistlines. And apparently these aren’t the only chips that can be too much of a good thing.

Just as too many potato chips aren’t good for you, too many ChIP results may lead us astray.

Chromatin immunoprecipitation (ChIP) is an incredibly valuable technique that lets us see where a particular protein binds in a genome. It can show us the target genes of a particular transcription factor, the distribution of RNA polymerases as they transcribe genes, the places where silencing proteins bind to turn off expression of particular regions, and lots more.

But just like potato chips, more ChIP results aren’t always better. Teytelman and coworkers, publishing in Proceedings of the National Academy of Sciences, and Park and coworkers, publishing in PLoS ONE, have discovered that highly transcribed regions of the genome consistently give false positive ChIP results. In other words, very active regions of the genome look like everything is binding there even when it almost certainly is not.  Teytelman and colleagues call these regions “hyper-ChIPable”. 

Far from being a reason to despair, though, the discovery of this artifact explains some puzzling previous results and inspires the creation of new, more reliable ChIP methods. This is exactly what Kasinathan and coworkers have done, in a recently published paper in Nature Methods. 

The idea behind the ChIP technique is that if you want to know all of the places across the genome where your protein of interest binds, you can lyse cells, shear the DNA into relatively short fragments, and immunoprecipitate your protein from the mixture. Usually the protein and DNA are cross-linked before immunoprecipitation, to strengthen their bond during the rest of the procedure.

After immunoprecipitation, the DNA fragments associated with the protein can be identified using a variety of methods. Finally, mapping the sequences of the fragments to the genomic sequence shows us all the sites that the protein occupies.

Teytelman and colleagues used ChIP-seq to ask whether the silencing complex (Sir2p, Sir3p, and Sir4p) ever binds to non-silenced regions of the genome. They thought they might see some binding, but they were astounded to find significant binding of the complex at 238 distinct euchromatic (non-silenced) loci. This didn’t really make sense, since the yeast Sir proteins are extremely well-studied and there were no biological hints that they have such a large presence at non-silenced genes. 

As a control, they looked at previously published ChIP data on the locations of two unrelated proteins, Ste12p and Cse4p, and found that their binding was enriched at the same 238 loci. Finally, they did a ChIP study using green fluorescent protein (GFP) alone. Sure enough, the ChIP data showed that this jellyfish protein apparently bound strongly to chromatin at those 238 sites! The common denominator shared by these loci: they were all very highly expressed.

Meanwhile, Park and coworkers were embarking on a similar journey. They found using ChIP-seq that several unrelated transcription factors seemed to have common targets, which didn’t make biological sense. Control experiments looking at binding sites of Mnn10p (a cytoplasmic protein not expected to have any contact with DNA), or even using nonspecific antibodies that didn’t recognize any yeast proteins, still gave the same set of ChIP targets. Again, these targets were all highly expressed genes.

Each group found several factors contributing to this artifact, although all the reasons why highly expressed regions yield false positives may not yet be uncovered.  But whatever the reasons, this finding helps explain some previously perplexing results – such as binding of Mediator complex all over the genome, or the paradoxical binding of silencing regulator Sir3p to the GAL1-GAL10 regulatory region under conditions where transcription is activated, not silenced.

In response to these issues, many researchers are actively trying to improve the ChIP technique. Kasinathan and colleagues have devised a method that they call ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) that eliminates crosslinking and substitutes micrococcal nuclease treatment for sonication (to shorten the DNA fragments).  In a pilot project, they mapped binding sites for the transcription factors Reb1p and Abf1p. The method looks to be both accurate and sensitive. Most binding locations that they found contained the binding motif sequence for that transcription factor, and also correlated with in vivo occupancy as determined by Dnase I footprinting – both of which support their biological relevance. Importantly, the technique shows no bias towards highly expressed regions.

The lesson for researchers is that ChIP results for highly expressed genes, particularly those done using older protocols, need to be viewed cautiously.  And of course this artifact could be an issue for organisms other than yeast. ChIP experiments are used across species, and have been valuable in elucidating the targets of disease-related proteins like the tumor suppressor p53.

The fact that yeast genetics and molecular biology have so well established the roles of certain chromatin-associated proteins was a key part of this puzzle, helping to point out the artifactual nature of some of the ChIP results. Just as a new recipe for potato chips could allow us to eat more of them while staying healthy, yeast research has led the way to a new recipe for more accurate ChIP studies.

Aside from the molecular biology behind this work, it is quite interesting from a sociological point of view as well. What is it like to make a discovery that calls into question a routinely-used technique and a lot of published results? Lenny Teytelman’s blog post on this topic provides a fascinating glimpse into this situation.