Sitting on the DNA, watching the tide roll away

Watching the chIPs roll in,

then I watch them roll away again,

I'm just sitting on the DNA,

wasting time

(sung to the tune of "Sitting on the dock of the bay" by Otis Redding)

Hesselberth et.al. recently published a paper about digital genomic
footprinting that blew me away because it has so much potential. The authors used DNAse I and Next Generation DNA Sequencing to map every site in the yeast genome where a protein might be sitting.

Since I used to do similar kinds of experiments, albeit on a much, much smaller scale, this sort of publication boggles my mind. It's only recently that I've come to terms with techniques like chIP and chIP Seq, and now, I imagine both of these will likely be replaced by this new method.

I'll be the first to admit that

it took some time for me to get used to the Next Generation DNA sequencing technologies and their potential to transform research, but now that I have, I'm hooked.

Hesselberth's paper describes a technique, developed and tested with yeast, that I find great because this method will allow us to ask two new kinds of questions, besides the one described in the paper.

We can now:

1.  Find all unknown genes  (because a transcription factor is sitting nearby).

2.  Combine the data from digital genomic footprinting with data from gene expression assays like RNA-Seq to provide a reality check for our RNA seq data.  For example, if we find an expression level in RNA-Seq of 10 transcripts per million, we'll be able to tell if that result is just background or a gene is just expressed at a very low level.

If we know a transcription factor is hanging out around our promoter, it would give some perspective to our RNA-Seq data.

Yes, I know for some of you this is complete gibberish, I'll do my best to explain some of it in English, and I might succeed or you might have to wait for later posts.

What is digital genomic footprinting?
I drew a picture to show what's happening in this technique.

Figure 1. Digital genomic footprinting, copyright® SGP

Chromosomal DNA is treated with DNAseI (yes, it looks like pac man in my drawing, so be it). DNAseI chews up all the DNA except, some DNA is protected because proteins are sitting there and blocking access. Then, the reaction is stopped and all the proteins are stripped off. Voila! We have short DNA fragments. Sequencing adaptors are added to the DNA fragments and they are sequenced. The reads that we get from the sequencing are aligned to the genome.

This is different from a ChIP seq or ChIP analysis because ChIP analysis requires us to have antibodies that will bind to the proteins and make the DNA-protein complexes precipitate.

What did the researchers find?
1.  The positions where proteins sit does correlate with earlier experiments, but the consensus motifs differed a bit from those found with chIP studies.

2.  They found some binding sites that had been missed with chIP.

3. The positions of fragments fit the known information about nucleotide accessibility.

4. They could get information about chromatin architecture.

In all, digital genomic footprinting is going to be a very powerful technique for gene prediction, annotation, and will complement data from expression analysis.  It will be a great benefit to uncover genes and promoters without having to know the sequence ahead of time or have antibodies on hand.

Good days are ahead for the genome world.

References

Hesselberth, J., Chen, X., Zhang, Z., Sabo, P., Sandstrom, R., Reynolds, A., Thurman, R., Neph, S., Kuehn, M., Noble, W., Fields, S., & Stamatoyannopoulos, J. (2009). Global mapping of protein-DNA interactions in vivo by digital genomic footprinting Nature Methods DOI: 10.1038/NMETH.1313