DNA loops the loop

Enhancers activate genes at long distance irrespective of position and orientation, so why don’t enhancers activate the wrong genes? The loop domain model proposes that DNA looping by insulators can restrict the reach of enhancers by generating isolated topological ‘DNA loop domains’. However, a biophysical understanding of this model has been lacking. By using well-characterised DNA looping proteins (Lac and Lambda CI repressors) in E. coli, we show that different loop arrangements can result in DNA looping assistance, loop interference or independent loops.

HFSP Program Grant holders Keith Shearwin and David Dunlap and colleagues
authored on Mon, 24 November 2014

Genes are frequently regulated by interactions between proteins that bind to the DNA near the gene and proteins that bind to DNA sites located far away, with the intervening DNA looped out. In eukaryotic genomes, genes and their distant sites are intermingled in complex ways and it is not understood how the correct connections are formed. Models that posit other DNA loops - that aid or inhibit enhancer to promoter contact - are difficult to test or quantitate rigorously in eukaryotic cells. Instead, we used the simple prokaryotic proteins Lac repressor and phage lambda CI (whose DNA looping properties we had characterized individually in an earlier paper (Priest et al., 2014)) to measure interactions between pairs of long DNA loops in E. coli cells, in the three possible topological arrangements (Figure 1). Using two pairs of DNA-looping sites, we tested the idea that one DNA loop can either assist or interfere with the formation of another DNA loop. By measuring the strength of these interactions between loops, we showed that this mechanism is capable of directing a distant site to the correct gene and preventing it contacting the wrong gene.

Figure 1

Nested loops assist each other’s formation, consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other’s formation, thus providing clear support for the loop domain model for insulation. Side-by-side loops do not affect each other. Mathematical modeling showed that combining loop assistance and loop interference can provide strong specificity in long-range interactions.  Figure 2 shows that a single DNA loop formed by the interaction of DNA-bound insulating proteins (I) can assist an enhancer (E) to contact one promoter (P), and simultaneously interfere with its ability to contact another promoter, thus achieving strong promoter-enhancer specificity.

Figure 2

We also used tethered particle motion (TPM) to detect and measure DNA looping in vitro. Looping and the associated assistance and interference effects were weaker in vitro than they were inside the cell. We suggest that DNA supercoiling, present in vivo, but absent in the TPM experiments could be a driving force behind the loop domain model.

Our results provide important support for the loop domain model and show that insulation is not restricted to complex regulatory elements in metazoan genomes but can occur by loop interference between relatively simple DNA-looping protein-binding sites.

Reference

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells. David G. Priest, Sandip Kumar, Yan Yan, David D. Dunlap, Ian B. Dodd, and Keith E. Shearwin. PNAS (2014) 111 (42) E4449-4457.

Other references

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors. David G. Priest, Lun Cui, Sandip Kumar, David D. Dunlap, Ian B. Dodd, and Keith E. Shearwin. PNAS (2014) 1111 (1) 349-354.

Link to PNAS article

Link to Pubmed