That’s why well-controlled experiments like those of the Stanford team are so useful, Kuhlman added. Usually, when researchers need to measure weak interactions like these, they have two choices: They can make a few very detailed, extremely precise measurements and generalize from them, or they can take a great many quick-and-dirty measurements and use mathematically complex statistical methods to deduce results. But Fordyce and her colleagues, Kuhlman said, used an automated, microfluidic chip-based procedure to take precise measurements during high-throughput experiments “to get the best of both worlds.”
The Stanford team found that different STR sequences can alter the binding affinities of transcription factors to DNA by as much as a factor of 70; they sometimes have more impact on transcription factor binding than changing the sequence of the binding motif itself. And the effects were different for the two different transcription factors they looked at.
So STRs seem able to fine-tune the ability of transcription factors to dock at a DNA site and thus to regulate a gene. But how, exactly?
A Waiting Room Near a Gene
The researchers figured that the part of a transcription factor that binds DNA might interact weakly with an STR, with the exact strength of that affinity depending on the STR sequence. Because such binding is feeble, it won’t have much specificity. But if a transcription factor is loosely grasped and released by an STR again and again, the cumulative effect is to keep the transcription factor in the vicinity of the gene so that it is more likely to bind securely to the motif region if needed.
Fordyce and her colleagues predicted that STRs thus act as a “lobby” or well where transcription factors can gather, however transiently, near a regulatory binding site. “The repetitive nature of an STR amplifies the weak effect of any single binding site that it is made of,” said Connor Horton, the first author on the study, who is now a doctoral student at the University of California, Berkeley.
Conversely, he added, some STRs can also act to pull transcription factors away from regulatory sequences, soaking up transcription factors elsewhere like a sponge. In this way, they can inhibit gene expression.
The work, Suter said, “shows convincingly that STRs directly impact binding of transcription factors in vitro.” What’s more, the Stanford team used a machine learning algorithm to show that the effects seen in their in vitro experiments also seem to be occurring in living cells (that is, in vivo).
But Robert Tjian, a biochemist at Berkeley and an investigator at the Howard Hughes Medical Institute, thinks it may be too early to be sure what influence a given STR-transcription factor combination has on gene expression in real cells.
Tjian, Xavier Darzacq and their colleagues in the lab they run together at Berkeley agree that STRs seem to offer a way of concentrating transcription factors near gene regulatory sites. Yet without knowing how close the factors need to be to activate transcription, it’s difficult to understand the functional significance of that result. Tjian said he would like to see whether introducing an STR into a living cell predictably influences the expression of a target gene. At present, he said, he is “not persuaded that STRs are necessarily going to be a major aspect of [regulatory] mechanisms in vivo.”
A Combinatorial Grammar
One lingering puzzle is how such a mechanism reliably provides the type of precise gene regulation that cells need, since both the strength and the selectivity of transcription factor binding within the STR wells are weak. Fordyce thinks that such specificity of influence could come from many sources — not just from differences in the STR sequences but also from cooperative interactions between transcription factors and other proteins involved in regulation.
Given all that, Horton said, it’s not clear that it will be straightforward to predict the effect of a given STR-transcription factor combination on the expression of a gene. The logic of the process is fuzzy indeed. And the “grammar” of the influence is probably combinatorial, Horton added: The outcome depends on different combinations of transcription factors and other molecules.
The Stanford team thinks that perhaps 90% of transcription factors are sensitive to STRs, but that there are many more types of transcription factors in the human genome than there are types of STRs. “Mutating an STR sequence might affect the binding of 20 different transcription factors in that cell type, leading to an overall decrease in transcription of that nearby gene without implicating any specific transcription factor,” Horton said.
So in effect, the Stanford team agrees with Tjian that gene regulation in living cells isn’t going to be driven by a single, simple mechanism. Rather, transcription factors, their DNA binding sites, and other regulatory molecules may assemble into dense gatherings that exert their influence collectively.
“There are now multiple examples that support the idea that DNA elements can crowd transcription factors to the point where they form condensates with cofactors,” said Richard Young, a cell biologist at the Whitehead Institute of the Massachusetts Institute of Technology. Enhancers bind many transcription factors to produce that crowding. STRs may be an ingredient that helps muster transcription factors to cluster near a gene, but they won’t be the whole story.
Why regulate genes in this complicated manner, rather than relying on the kind of strong and specific interactions between regulatory proteins and DNA sites that dominate in prokaryotes? It’s possible that such fuzziness is what made large complex metazoans possible at all.
To be viable species, organisms need to be able to evolve and adapt to changing circumstances. If our cells relied on some huge yet tightly prescribed network of gene regulatory interactions, it would be difficult to make any changes to it without disrupting the whole contraption, just as a Swiss watch will seize up if we remove (or even slightly displace) any of its myriad cogwheels. If the regulatory molecular interactions are loose and rather unspecific, however, there is useful slack in the system — just as a committee can generally come to a good decision even if one of its members is out sick.
Fordyce notes that in prokaryotes like bacteria, it may be relatively easy for transcription factors to find their binding sites because the genome to be searched is smaller. But that gets harder as the genome gets bigger. In the big genomes of eukaryotes, “you can no longer tolerate the risk that you will become transiently stuck at a ‘wrong’ binding site,” Fordyce said, because that would compromise the ability to respond quickly to changing environmental conditions.
Moreover, STRs themselves are highly evolvable. A lengthening or shortening of their sequence, or an alteration to the size and depth of the “transcription factor well,” can occur easily through mishaps in DNA replication or repair, or through sexual recombination of the chromosomes. To Fordyce, it suggests that STRs “may therefore serve as the raw material for evolving new regulatory elements and fine-tuning existing regulatory modules for sensitive transcriptional programs,” such as those governing the development of animals and plants.
The Power of Weak Interactions
Such considerations are leading molecular biologists to pay much more attention to weak and relatively unselective interactions in the genome. Many of these involve proteins that, instead of having a fixed and precise structure, are loose and floppy — “intrinsically disordered,” as biochemists put it. If proteins only worked through rigid structural domains, Young explained, it would constrain not only how well regulatory systems could evolve but also the kinds of dynamic regulation seen in life. “You won’t find a living organism — or even a virus — functioning with only stable structural elements like those in a Swiss watch,” Young said.
Perhaps evolution just stumbled on STRs as a component of such a complex but ultimately more effective solution to gene regulation in eukaryotes. STRs themselves may arise in several ways — for example, through errors in DNA replication or the activity of DNA segments called transposable elements that make copies of themselves throughout the genome.
“It just so happened that the resulting emergent weak interactions between proteins and the repetitive sequences was something that could … provide selective advantage to the cells where it occurred,” Kuhlman said. His guess is that this fuzziness was probably forced upon eukaryotes, but that “they were subsequently able to exploit [it] for their own benefit.” Bacteria and other prokaryotes can rely on well-defined “digital” regulatory logic because their cells tend to exist in only a few simple, distinct states, such as moving around and replicating.
But the different cell states for metazoans are “much more complex and sometimes close to a continuum,” Suter said, so they are better served by fuzzier “analog” regulation.
“The gene regulatory systems in bacteria and eukaryotes do seem to have diverged quite substantially,” Tjian agreed. While Monod is said to have once remarked that “what is true for E. coli is true for the elephant,” it seems that isn’t always so.
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