Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops-that aid or inhibit enhancerpromoter contact-are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. 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. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.Lac repressor | lambda CI | tethered particle motion | statistical mechanical modeling T ranscription of genes is regulated by promoter-proximal DNA elements and distal DNA elements that together determine condition-dependent gene expression. In eukaryotic genomes, enhancers can be many hundreds of kilobases away from the promoter they regulate (1-3), and the intervening DNA can contain other promoters and other enhancers (4-7). How the regulatory influence of distal elements is exerted efficiently and specifically at the correct promoters is poorly understood.Enhancers are clusters of binding sites for transcription factors and chromatin-modifying enzymes, and activate promoters by directly contacting them via DNA looping (8-12). Enhancer trap approaches and mapping of transcription factor binding and chromatin modifications have identified tens of thousands of enhancer elements in metazoan genomes (7,(13)(14)(15)(16). Chromatin capture studies show that enhancers and promoters are connected in highly complex condition-dependent patterns (6,15,17). Although core enhancer and promoter elements can provide some specificity (18), enhancers are often able to activate heterologous promoters if they are placed near to each other. Indeed, this lack of specificity is the basis for standard enhancer assays and screens (7,14,19). Thus, additional mechanisms are clearly needed to target enhancers to the correct promoters over long distances and to prevent their interaction with the wrong promoters. Dedicated DNA-looping elements that can either assist or interfere with enhancer-promoter looping are thought to play a major role.In theory, any DNA loop that brings the enhancer and promoter closer together should assist their interaction (Fig. 1A), because the efficiency of contact increases as the length of the DNA tether between the sites shortens (20-24). Promoter-tethering elements in Drosophila that allow activation by specific enhancers over long distances are proposed to form DNA loops between sequences near the ...

Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation, but to what extent supercoiling or nucleoid associated proteins promote looping is poorly understood. Using the lac repressor (LacI), a paradigmatic loop-mediating protein, we measured LacI-induced looping as a function of either supercoiling or the concentration of the HU protein, an abundant nucleoid protein in Escherichia coli. Negative supercoiling to physiological levels with magnetic tweezers easily drove the looping probability from 0 to 100% in single DNA molecules under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU could not raise the looping probability above 30% in similarly stretched DNA or 80% in DNA without tension. Negative supercoiling is required to induce significant looping of DNA under any appreciable tension.

Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites.

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop‐mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

Emerging evidence indicates that in addition to its well-recognized functions in antiviral RNA silencing, dsRNA elicits pattern-triggered immunity (PTI), likely contributing to plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multi-color in vivo imaging, analysis of green fluorescent protein (GFP) mobility, callose staining and plasmodesmal marker lines in Arabidopsis thaliana and Nicotiana benthamiana, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) the BOTRYTIS INDUCED KINASE1 (BIK1)/AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE KINASE1 (BIK1/PBL1) kinase module, PLASMODESMATA-LOCATED PROTEINs (PDLPs)1/2/3, as well as CALMODULIN-LIKE 41 (CML41) and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. Unlike the classical bacterial elicitor flagellin, dsRNA does not trigger detectable reactive oxygen species (ROS) burst, substantiating the idea that different microbial patterns trigger partially shared immune signaling frameworks with distinct features. Likely as a counter strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support a model in which plant immune signaling constrains virus movement by inducing callose deposition at plasmodesmata and reveals how viruses counteract this layer of immunity.