Concentration and Length Dependence of DNA Looping in Transcriptional Regulation

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: Discussionmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

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

Priest

Kumar²,

Yan³

et al. 2014

“…for K d by estimating the concentration at which the two peaks have equal weight (and thus, the concentration where either of these two states is equally likely). More formally, we used a statistical mechanical model of binding (19,20) to extract the relevant K d values taking into account the binding histograms over the entire concentration range. In this case, for the 12RSS site, we found K d = 13.9 ± 4.7 nM, which is within error of a previously reported value of 14.5 ± 2.4 nM (21) and less than the only other measured value of K d = 25.0 ± 5.0 nM derived from bulk experiments (22).…”

Section: Resultsmentioning

confidence: 99%

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Lovely

Brewster

Schatz

et al. 2015

Self Cite

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed singlemolecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites. The recombination-activating genes, RAG1 and RAG2, encode proteins that carry out V(D)J recombination by bringing a 12RSS and a 23RSS together into a paired (or synaptic) complex, nicking the RSS sites adjacent to the heptamer, and converting each nick into a double-strand break, leaving a hairpin at the coding end (Fig. 1). The hairpin is created by nucleophilic attack on the opposing strand by the 3′ hydroxyl group at the nick in a transesterification reaction. After RAG1/2 forms hairpins, it recruits the nonhomologous end joining machinery to repair the ends (1, 2). Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3, 4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5). High-mobility group-box protein 1 (HMGB1) acts as a cofactor and increases RAG1/2c affinity for the 23RSS (6). HMGB1 is required for paired complex formation and efficient conversion of RAG-mediated nicks into hairpins (7-10).Current in vitro assays that capture the paired complex with RAG1/2c generally place a 12RSS and a 23RSS on two different DNA molecules, typically short oligonucleotides. However, in vivo, antigen receptor loci are assembled using RSSs on the same DNA molecule (1, 11). One prior study captured RAG1/2c and HMGB1 bound to DNA with a 12RSS and a 23RSS on the same substrate using standard bulk assays (12). However, many key mechanistic questions remain unresolved concerning how a diverse immune repertoire is generated by the RAG recombinas...

“…Previous studies indicate that LacI looping with short DNA tethers (<500 bp) is less efficient in vitro than in vivo. To test whether this difference holds for longer DNA tethers, we examined LacI looping in single molecules by TPM (9,(30)(31)(32), where looping of a DNA molecule attaching a bead to a surface can be detected as a restriction in the Brownian motion of the bead (Fig. 5A).…”

Section: Effect Of Separation On Long-range Looping By λ Repressor Inmentioning

confidence: 99%

“…Measurements of j LOOP for protein-induced looping give a similar picture. Most studies have focused on linear tethers <500 bp, finding variable j LOOP values generally below 100 nM (9). Lower j LOOP values are seen at longer separations: ∼18 nM for Cre DNA recombination at 3,000 bp (15), whereas LacI DNA binding cooperativity was undetectable at Significance Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression.…”

mentioning

confidence: 99%

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Priest

Cui

Kumar

et al. 2013

Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to longrange DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250-to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5-to 10-kb distances, was able to increase contact probability 10-to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5-to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells. j factor | TPM | promoter-enhancer I nteractions between proteins bound to separate sites on the same DNA molecule are critical in gene regulation and other DNA processes (1-4). The DNA separation between functionally interacting sites ranges from a few base pairs to hundreds of kilobase pairs, as in the case of some eukaryotic enhancers and their promoters. At short separations, it is clear that the DNA acts as a tether that keeps one site in the vicinity of the other so that the proteins at one site can find the other site in 3D space more efficiently than if they were free in solution (Fig. 1). Tethering is also a way to provide specificity because it aids interaction with linked sites but not unlinked sites. However, as the separation between the sites increases, this tethering effect becomes weaker, and it is not understood how the DNA linkage between widely separated sites, for example, between enhancers and promoters, provides the efficiency and specificity required for proper regulation.The effect of DNA tethering can be quantified by the factor j LOOP (M), the effective molar concentration of one site on the DNA relative to the other, or as the free energy of the DNA looping reaction ΔG LOOP (Fig. 1) (5-9). These parameters are interconvertible: ΔG LOOP = -RTlnj LOOP (kcal/mol; the reference j is 1 M). The formation of a naked DNA loop is in itself an energetically unfavorable reaction (ΔG LOOP is positive) under physiological conditions due to the enthalpic cost of DNA bending and twisting (particularly important for s...