Bacterial Repression Loops Require Enhanced DNA Flexibility

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 ...

Section: Resultsmentioning

confidence: 79%

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

Priest

Kumar²,

Yan³

et al. 2014

“…Our data allow one calculating average bending rigidity and average helical repeat for a DNA segment with any chosen sequence. These average values can be used to evaluate the sequence-dependent energy of DNA deformation in short loops which are involved in transcription regulation (37)(38)(39). In such loops, where DNA is constrained at its ends only, the double helix fluctuates around the conformation with the minimum energy of elastic deformation, similar to the situation in the minicircles used in this study.…”

Section: Discussionmentioning

confidence: 88%

Sequence dependence of DNA bending rigidity

Geggier

Vologodskii

2010

212

247

For many aspects of DNA-protein interaction, it is vital to know how DNA bending rigidity (or persistence length, a) depends on its sequence. We addressed this problem using the method based on cyclization of short DNA fragments, which allows very accurate determination of a. Our approach was based on assigning specific values of a to each of 10 distinct dinucleotide steps. We prepared DNA fragments, each about 200 bp in length, with various quasiperiodic sequences, measured their cyclization efficiencies (j factors), and fitted the data by the theoretical equation to obtain the values of a for each fragment. From these data, we obtained a set of a for the dinucleotide steps. To test this set, we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments, and determined their values of a experimentally. The measured and calculated values of a were very close to one another, confirming that we have found the correct solution to this long-standing problem. The same experimental data also allowed us to determine the sequence dependence of DNA helical repeat.DNA elasticity | DNA persistence length T he value of DNA persistence length, a, closely related to the bending rigidity of the double helix, is very important for quantitative analysis of many aspects of DNA functioning. Many different methods were applied over the last decades to determine this value and its dependence on ionic conditions (1). These studies showed that under near physiological ionic conditions the value of a is close to 50 nm (1-3). They also showed that, in a good approximation, the value of a does not depend on DNA sequence, because many studies that use different DNA molecules gave very close values of a. However, the sequence dependence of DNA bending rigidity is vital for many aspects of DNAprotein interaction, and is particularly important for understanding how nucleosomes position along DNA molecules (4, 5). Despite its importance, the problem has formerly remained unsolved, although a few research groups have tried various approaches (6-8). The data that are generally considered most reliable were obtained from statistical analysis of DNA-protein crystal structures (9, 10). The large volume of available structural data allowed this group of researchers to present a very detailed picture of the sequence dependence of DNA conformational properties. This analysis, however, is based on the assumption that variations of the DNA bend angles in the crystals correspond to the amplitudes of thermal fluctuations of these angles in solution. It is hard to justify this assumption.The major obstacle in determining the sequence dependence of a is the necessity of highly accurate measurements of a for different sequences. The majority of methods applied for the measurements of a do not provide the necessary level of accuracy. The only known method that allows one to measure a with the required accuracy is based on cyclization of short DNA fragments by DNA ligase (2,3,11,12). It i...

“…Linear DNA segments of 50-180 bp appear to loop with the extended LacI conformation: DNaseI footprinting (loops of 52 and 74 bp) and gel-mobility patterns (loops of 153, 158, 163, and 168 bp) (19) are best interpreted assuming P1 E configuration (9). Loops in the size range of 65-90 bp adopt either P1 or P1 E structure, depending on whether HU is, respectively, present or not (20). This difference was attributed to the tendency of HU to induce the sharp bending of DNA required for P1.…”

mentioning

confidence: 99%

Tetramer opening in LacI-mediated DNA looping

Rutkauskas

Zhan²,

Matthews

et al. 2009

Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations. (1). This effect is due to the fact that LacI is a homotetrameric protein assembled as a dimer of dimers, with each dimer presenting a DNA-binding domain, enabling the tetramer to bind simultaneously to two operators (2) and form a loop in the intervening DNA ( Fig. 1 A and B). Consequently, LacI binding to one of the auxiliary operators increases the proximity of the remaining free DNA-binding domain to the primary operator, thus enhancing the probability of transcription blocking. Moreover, upon dissociation from the primary operator, the repressor is likely to remain bound to one of the auxiliary operators, avoiding diffusion into the cytosol and retaining proximity to the primary operator to facilitate rebinding. Thus, LacI-mediated DNA looping is fundamental for the efficiency of repression, as demonstrated by the drastic effects of deletion of auxiliary operators (3).In general, DNA looping is a recurring motif among several other DNA-binding proteins with different functions (4). The looping probability is quantified by the J m factor (5), defined as the local concentration of the LacI bound to one of the operators with respect to the remaining free operator. This factor is governed by the DNA-protein mechanics, as manifest in the profound dependence of in vivo repression (6) or in vitro cyclization efficiency (7) on interoperator spacing. Modulation of this property with the period of helical repeat is due to a large DNA-twisting-associated energetic penalty and, therefore, certain phase requirements for joining the loose DNA ends for cycliz...