Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions 1 . However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA – a proxy for DNA bendability – in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors 2 by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a ‘mechanical code’ with broad functional implications.

Mechanical deformations of DNA such as bending are ubiquitous and implicated in diverse cellular functions. However, the lack of high-throughput tools to directly measure the mechanical properties of DNA limits our understanding of whether and how DNA sequences modulate DNA mechanics and associated chromatin transactions genome-wide. We developed an assay called loop-seq to measure the intrinsic cyclizability of DNA, a proxy for DNA bendability, in high throughput. We measured the intrinsic cyclizabilities of 270,806 50 bp DNA fragments that span the entire length of S. cerevisiae chromosome V and other genomic regions, and also include random sequences. We discovered sequence-encoded regions of unusually low bendability upstream of Transcription Start Sites (TSSs). These regions disfavor the sharp DNA bending required for nucleosome formation and are co-centric with known Nucleosome Depleted Regions (NDRs). We show biochemically that low bendability of linker DNA located about 40 bp away from a nucleosome edge inhibits nucleosome sliding into the linker by the chromatin remodeler INO80. The observation explains how INO80 can create promoter-proximal nucleosomal arrays in the absence of any other factors by reading the DNA mechanical landscape. We show that chromosome wide, nucleosomes are characterized by high DNA bendability near dyads and low bendability near the linkers. This contrast increases for nucleosomes deeper into gene bodies, suggesting that DNA mechanics plays a previously unappreciated role in organizing nucleosomes far from the TSS, where nucleosome remodelers predominate. Importantly, random substitution of synonymous codons does not preserve this contrast, suggesting that the evolution of codon choice has been impacted by selective pressure to preserve sequence-encoded mechanical modulations along genes. We also provide evidence that transcription through the TSS-proximal nucleosomes is impacted by local DNA mechanics. Overall, this first genome-scale map of DNA mechanics hints at a mechanical code with broad functional implications.

Sequence features have long been known to influence the local mechanical properties and shapes of DNA. However, a mechanical code (i.e. a comprehensive mapping between DNA sequence and mechanical properties), if it exists, has been difficult to experimentally determine because direct means of measuring the mechanical properties of DNA are typically limited in throughput. Here we use Loop-seq – a recently developed technique to measure the intrinsic cyclizabilities (a proxy for bendability) of DNA fragments in genomic-scale throughput – to characterize the mechanical code. We tabulate how DNA sequence features (distribution patterns of all possible dinucleotides and dinucleotide pairs) influence intrinsic cyclizability, and build a linear model to predict intrinsic cyclizability from sequence. Using our model, we predict that DNA mechanical landscape shapes nucleosome organization around the promoters of various organisms and at the binding site of the transcription factor CTCF, and that hyperperiodic DNA in C. elegans leads to globally curved DNA segments. By performing loop-seq on random libraries in the presence or absence of CpG methylation, we show that CpG methylation leads to global stiffening of DNA in a wide sequence context, and predict based on our model that CpG methylation widely changes the mechanical landscape around mouse promoters. It suggests how epigenetic modifications of DNA might alter gene expression and mediate cellular adaptation by affecting critical processes around promoters that require mechanical deformations of DNA, such as nucleosome organization and transcription initiation. Finally, we show that the genetic code and the mechanical code are linked: sequence-dependent mechanical properties of coding DNA constrains the amino acid sequence despite the degeneracy in the genetic code. Our measurements explain why the pattern of nucleosome organization along genes influences the distribution of amino acids in the translated polypeptide.

Chronic osteoarthritis (OA) is a widespread source of pain and disability and represents a growing economic burden across aging populations. Representing a major focus of arthritis care, OA of the knee is especially concerning as it has the potential to restrict mobility and significantly impair quality of life. Chronic OA is often poorly managed both pharmacologically and nonpharmacologically, with surgical management representing the definitive treatment. Those who are not surgical candidates or simply opt for minimally invasive treatments are usually faced with a lack of alternatives. An additional treatment presents itself in the form of water-cooled radiofrequency ablation, which involves the use of thermal lesions to interrupt the active pain pathways. An 81-year-old woman with bilateral severe knee OA was initially seen and evaluated in an outpatient physiatry clinic after multiple previous workups of her ongoing knee pain. With a known diagnosis of end-stage knee OA, the patient chose to proceed with bilateral water-cooled radiofrequency ablation. At 6 weeks and 3 months after the procedure, the patient maintained adequate levels of pain relief, markedly improved function, and enhanced quality of life. Water-cooled radiofrequency ablation has the potential to create lasting pain relief and with minimal adverse effects in patients with chronic knee OA.

Diverse DNA-deforming processes are impacted by the local mechanical and structural properties of DNA, which in turn depend on local sequence and epigenetic modifications. Deciphering this 'mechanical code', i.e., this dependence, has been challenging due to the lack of high-throughput experimental methods. Here we present a comprehensive characterization of the mechanical code. Utilizing high-throughput measurements of DNA bendability via loop-seq, we quantitatively established how the occurrence and spatial distribution of dinucleotide, tetranucleotides, and methylated CpG, impact DNA bendability. We use our measurements to develop a physical model for the sequence and methylation dependence of DNA bendability. We validate the model by performing loop-seq on mouse genomic sequences around transcription start sites and CTCF binding sites. We apply our model to test the predictions of all atom molecular dynamics simulations, and to demonstrate that sequence and epigenetic modifications can mechanically encode regulatory information in diverse contexts.