The "duon" delusion and why transcription factors MUST bind non-functionally to exon sequences

This post is about a paper recently published in Science (Dec. 13, 2013) by John Stamatoyannopoulos and his collaborators at the University of Washington in Seattle, Washington, USA.

Stergachis, A.B., Haugen, E., Shafer, A., Fu, W., Vernot, B., Reynolds, A., Raubitschek, A., Ziegler, S., LeProust, E.M., Akey, J.M. and Stamatoyannopoulos, J.A. (2013) Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution. Science 342:1367-1372. [doi: 10.1126/science.1243490] [Abstract] [PDF]

Stamatoyannopoulos is one of the ENCODE workers. He recently gave a talk at the University of Toronto where he defended the idea that pervasive transcription and pervasive transcription factor binding are evidence of widespread function in the human genome. This paper looks at transcription factor binding sites in exon sequences (coding sequences) and finds lots of them. What this means is that stretches of coding region contain codons AND transcription factor binding sites (duh!).

This is such an important discovery (not!) that Stergachis et al. coined a new word, "duons," to describe sequences that have two meanings. The ridiculous hype over this paper is covered in a separate post []. Here, I want to look at the science.

Let's start by reviewing what we know about DNA binding proteins. Some of these proteins bind to specific sequences in DNA. The classic examples are the restriction enzymes (restriction endonucleases) produced by various bacterial species to protect themselves against invasion by foreign DNA. These enzymes recognize short sequences of DNA. They bind and cleave the DNA by cutting both strands [see Restriction, Modification, and Epigenetics].

Typical DNA binding proteins recognize specific sequences of about six base pairs. The restriction enzyme EcoR1, for example, binds to the sequence GAATTC. This sequence will occur quite often in any random stretch of DNA. You can calculate the frequency by determining the probability of GAATTC: it's 4⁶ or one in 4096 base pairs. What this means is that EcoR1 will bind to any DNA about once every 4000 bp (4Kb).¹

Back in the olden days, before DNA sequencing became cheap and easy, we used to construct restriction maps of DNA to define genes. Here's an example from a paper we published over thirty years ago. It shows the DNA binding sites of various restriction enzymes on Drosophila melanogaster DNA clones containing hsp70 heat shock genes (Moran et al. 1979).

The important point here is that none of these bacterial enzymes will ever see Drosophila DNA outside of the laboratory but because of their binding properties they recognize their specific binding sequence whenever they encounter it. I could have done the same experiment using transcription factors. If I had several dozen transcription factors from human cells, I could have mapped their binding sites on my Drosophila DNA and made a figure just like the one shown above. Of course, none of those binding sites would be biologically relevant since the binding of a human transcription factor to fruit fly DNA isn't ever gong to happen in the real world.

The coding regions of the genes are shown by the solid black bars in the figure. (These genes have no introns.) Note that the restriction enzyme binding sites are distributed fairly randomly but many of them bind to the coding region. What this means is that certain sequences in the coding region have a dual "meaning." Not only do they specify codons, they also specify the binding site for a restriction endonuclease. I suppose we could have made a big deal of this back in 1979 and called those sequences "duons" but I doubt very much this would have got past the reviewers. It's too obvious and it's not biologically relevant.

Same with transcription binding sites. If I had published a map of human transcription factor binding sites on Drosophila DNA nobody would think this remarkable enough to coin a new word for codons that are also transcription factor binding sites.

This brings us back to the Stergachis et al. (2013) paper. What they did was to map the sites in human DNA that bind human transcription factors. Recall that there have to be lots of these sites in our genome since most of them recognize specific sequences of about 6bp. Like restriction enzyme binding sites, these sequences will occur once every 4Kb (4000 bp) in random sequences of DNA. The human genome is not exactly random DNA but it's close enough for our purposes. For any given transcription factor, there will be about 800,000 binding sites in the human genome. Only a tiny percentage of these sites will be be biologically relevant leading to regular transcription of a nearby gene.

Stergachis et al. did not map transcription factor binding sites to naked human DNA. Instead, they mapped the binding sites in vivo which means that a given cell type had to produce the transcription factor and the DNA binding site had to be accessible. The latter distinction is important because a lot of our DNA is tightly bound to nucleosomes to make chromatin and in higher order chromatin structures the naked DNA is not "visible" to DNA binding proteins. When a gene is active, the chromatin opens up to form an "open" chromatin region where the DNA is exposed to transcription factors and RNA polymerase transcription complexes.

What this means is that only a subset of possible transcription factor binding sites can be detected in any given cell type. Many of these will be in or near active genes where the chromatin is in an "open" conformation. This includes protein coding genes and coding regions.

Stergachis et al. (2013) found a total of 11,588,043 transcription factor binding sites in 81 different cell types. The average was 1,018,514 different binding sites in a typical cell. They found a total of 24,842 binding sites within protein-coding exons. This corresponds to 1.8% of the total. In other words, 98.2% of the binding sites were in noncoding DNA and 1.8% were in coding DNA. This is pretty close to the distribution of coding and noncoding DNA in the genome suggesting strongly that the method is detecting random non-functional binding of transcription factors.

I conclude that the authors are detecting transcription factors binding to non-functional sites within coding regions. The vast majority of these sites have no biological relevance. They simply reflect the occurrence of fortuitous binding sites in the genome that just happen to match the specific binding site consensus sequence. This is not a big deal. In fact, it is predicted simply on the basis of our understanding of DNA binding proteins.

The authors do not address this possibility in their paper. Instead, they conclude ...

Our results indicate that simultaneous encoding of amino acid and regulatory information within exons is a major functional feature of complex genomes. The information architecture of the received genetic code is optimized for super-imposition of additional information, and this intrinsic flexibility has been extensively exploited by natural selection. Although TF binding within exons may serve multiple functional roles, our analyses above is agnostic to these roles, which may be complex.

While the authors are entitled to their opinion, they are NOT entitled to ignore other possible interpretations of their data. Especially since, in this case, the other interpretation contradicts the main conclusions of the paper. This is not how science should be done. This paper should never have been published as it is. The reviewers should be named and shamed. The editor(s) at Science should be fired.²

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1. It's actually a bit more complicated than that. Most DNA binding proteins bind nonspecifically to any piece of DNA. It's not just part of the intrinsic affinity for a negatively charged double-helix, it's also biologically relevant since these proteins usually bind weakly to DNA and then scan the DNA in one dimension looking for their specific binding site [see Slip Slidin' Along - How DNA Binding Proteins Find Their Target]. This kind of binding would not show up in the methods used by Stergachis et al. However, specific DNA binding proteins will recognize sequences that are closely related, but not identical, to their binding site and these interactions could be detected. EcoR1, for example, will bind with appreciable affinity to sites that differ by just one base pair from GAATTC.

2. No editor at Science should be unaware of the controversy surrounding the ENCODE publicity fiasco of 2012. Thus, editors should be on high alert every time they receive a paper from another ENCODE lab. They should go out of their way to choose reviewers who have been critical of the claims of pervasive functionality.

[Image Credit: Moran, L.A., Horton, H.R., Scrimgeour, K.G., and Perry, M.D. (2012) Principles of Biochemistry 5th ed., Pearson Education Inc. page 581 [Pearson: Principles of Biochemistry 5/E] © 2012 Pearson Education Inc.]

Moran, L., Mirault, M.-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W.J. (1979) Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

Stergachis, A.B., Haugen, E., Shafer, A., Fu, W., Vernot, B., Reynolds, A., Raubitschek, A., Ziegler, S., LeProust, E.M., Akey, J.M. and Stamatoyannopoulos, J.A. (2013) Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution. Science 342:1367-1372. [doi: 10.1126/science.1243490] [Abstract] [PDF]