Abstract
Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.
Highlights
It has been known since the work of Jacob and Monod that genomes encode special regulatory sequences in the form of binding sites for proteins that modulate transcription, only recently has it become clear that genomes encode other regulatory features in their sequences as well
We compare the effects on looping of this poly(dA:dT)-rich DNA to the effects of two synthetic sequences we have previously studied, and to those of two additional naturally occurring, genomic sequences: the well-known, strong nucleosome positioning sequence 5S from a sea urchin ribosomal subunit [45], which, along with the 601TA sequence we previously studied, contains the repeating AA/TT/TA/AT and offset GG/CC/CG/ GC steps that are common in nucleosome-preferring sequences; and one of the GC-rich sequences that are abundant in the exons and regulatory regions of human genes, and that correlate with high nucleosome occupancy in vivo [18,19,22]
As shown schematically in Fig. 1(A), in tethered particle motion (TPM), a microscopic bead is tethered to a microscope coverslip by a linear piece of DNA, with the motion of the bead serving as a reporter of the state of the DNA tether: the formation of a protein-mediated DNA loop in the tether reduces the motion of the bead in a detectable fashion [47,48,49,50]
Summary
It has been known since the work of Jacob and Monod that genomes encode special regulatory sequences in the form of binding sites for proteins that modulate transcription, only recently has it become clear that genomes encode other regulatory features in their sequences as well. One well-known example of other information present in genomes is the different sequence preferences that confer nucleosome positioning [1,2,3], with similar ideas at least partially relevant in the context of architectural proteins in bacteria [4] It has been shown both from analyses of sequences isolated from natural sources and from in vitro nucleosome affinity studies with synthetic sequences that the DNA sequence can cause the relative affinity of nucleosomes for DNA to vary over several orders of magnitude, most likely due to the intrinsic flexibility, especially bendability, of the particular DNA sequence in question [3,5,6,7,8]. Our aim here is to explore the extent to which these sequences, when taken beyond the context of cyclization and nucleosome formation to another critical DNA deformation motif, exhibit similar effects on a distinct kind of deformation
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