An approach to monitor genomic interactions in live cells

In previous studies we tracked either V H or D H J H motion in live pro-B cells using tandem arrays of TET-operator binding sites42. We next aimed to monitor V H -D H J H encounters directly in live cells using a dual labeling approach (Fig. 1a). Attempts to insert tandem arrays of LAC operators in conjunction with TET-operator binding sites failed because tandem arrays of LAC operator binding sites were unstable in expanding pro-B cell cultures. We therefore developed a strategy to simultaneously track the motion of paired genomic elements. Specifically, we used wild-type and mutant TET-repressor binding sites associated with unique DNA binding specificities45,46. From these, we selected a mutant TET repressor (4C5G) carrying two amino acid substitutions (E37A and P39K) in the TET-DNA binding domain (Fig. 1b). Tandem arrays containing 360 copies of the mutant TET 4C5G binding site (CCGTCAGTGACGG) were constructed essentially as described previously (Fig. 1c)47.

In principle, two distinct repressor modules should permit the simultaneous tracking of two genomic regions. However, heterodimers involving wild-type TET and mutant TET would readily be assembled interfering with their ability to selectively bind their cognate homo-dimeric binding sites. To overcome this obstacle, we converted the MUT-TET repressor dimer into a single chain using a 29 amino acid linker. The single chain (sc) TET-repressor binds to its cognate binding site as a monomer45. To generate two differentially labeled fusion proteins, the wild-type TET-repressor was fused to GFP while the mutant (sc) TET (4C5G) repressor was tagged with a polypeptide (SNAP-TAG) that binds cell-permeable fluorescent dyes (Fig. 1d). To boost expression of the wild-type and mutant TET-repressors, both proteins were codon-optimized. Finally, to optimize the signal-to-background fluorescence ratio, the TET-SNAP-tag repressor was placed under control of a mutant Kozak sequence (GCCTCCATG) to dampen translation and reduce background fluorescence (Fig. 1d).

Tracking V H -D H J H motion in live B-lymphocytes

In previous studies, we generated mice that carried tandem arrays of WT-TET operator binding sites in either the V H or D H J H regions43. From these mice we derived immortalized RAG-deficient pro-B cell lines. Briefly, RAG2-deficient WT-TET D H J H pro-B cells were isolated, grown in the presence of IL7 and SCF and transduced with BCR-ABL virus. This approach yielded BCR-ABL-transformed pro-B cell lines that carried a tandem array of TET-operator binding sites adjacent to the D H J H region (Fig. 1a). Next, using CAS9-CRISPR engineering a tandem array of mutant TET repressor binding sites was inserted into the V H 8-3 region (Supplementary Fig. 1). Cells that harbored the array of MUT-TET binding sites in the V H 8-3 region were expanded and transduced with virus expressing WT-TET GFP and MUT-TET SNAP-TAG. Two days post viral transduction the cells were incubated with the cell-permeable dye TMR-STAR to label MUT-TET bound sites and imaged using the OMX microscope platform. Cells transduced with virus expressing WT-GFP revealed two fluorescent foci consistent with WT-TET binding near the D H J H region at both alleles (Fig. 2a; left panels). Cells transduced with virus expressing MUT-TET SNAP TAG revealed a single fluorescent center consistent with the insertion of MUT-TET binding sites on one allele (Fig. 2a, middle panels). Cells transduced with virus expressing WT-TET GFP and MUT-TET SNAP TAG showed one allele marked for both the V H and D H J H regions and another allele marked with only the D H J H region (Fig. 2a, right panels). Transduced V H MUT-TET; D H J H WT-TET pro-B cells were imaged every 2 s for 400 s or every 40 s for 4800 s. We found that, depending on the efficiency of viral transduction, the V H and D H J H region were marked by both red and green fluorescence in 1–10% of the transduced pro-B cell population.

Fig. 2 Tracking V H -D H J H motion in pro-B cells. a Live imaging of pro-B cells harboring D H J H WT-TET and V H MUT-TET alleles. Panels indicate pro-B cells carrying D H J H WT-TET and V H MUT-TET alleles transduced with WT-TET GFP and MUT-TET SNAP-TAG. Left panels display pro-B cells transduced with virus expressing WT-GEP. Middle panels show pro-B cells transduced with virus expressing MUT-TET SNAP-TAG. Right panels show pro-B cells transduced with WT-GFP and MUT-TET SNAP-TAG. Scale bar corresponds to 2 μm. b Left panels show pro-B cell lines harboring tandem arrays of WT-TET GFP and MUT-TET SNAP-TAG analyzed using 3D-FISH. Fluorescently labeled V H and D H J H probes were used for visualization. Middle panels show fluorescent foci obtained from live-cell imaging. Right panel shows the probability distributions of spatial distances separating the V H and D H J H elements obtained from live pro-B cell imaging (black dots) compared to those obtained from 3D-FISH measurements (orange dots). Numbers of cells that were analyzed is indicated. c Experimental strategy is displayed to determine localization error. Localization error was determined by measuring the spatial distances separating red and green fluorescent signals. Fifty cells were analyzed. Scale bars represents 10 μm. Source data are provided as a Source Data File Full size image

To validate this approach, we compared the distribution of the spatial distances separating the V H and D H J H elements obtained from live-cell imaging to those obtained from three- dimensional (3D)-FISH measurements (Fig. 2b). We found that both methods yielded similar distributions, consistent with the insertion of tandem arrays of WT-TET and MUT-TET binding sites into the V H and D H J H regions (Fig. 2b, right panel). To measure localization error, pro-B cells harboring tandem arrays of MUT-TET binding sites were transduced with virus expressing MUT-TET GFP (green fluorescence) and virus expressing MUT-TET SNAP-TAG (red fluorescence) (Fig. 2c). Cells that showed overlapping green and red fluorescent foci were imaged every 2 s for 400 s. The mean-squared displacement was ~0.02 µm2, i.e., an average localization error of 22 nm for the x- and y-coordinates and 95 nm for the z-coordinate for each marker. To extract the intrinsic V H -D H J H genomic motion from the observed motion, rotational, and translational nuclear motion, as well as localization error were eliminated using two independent procedures (Supplementary Fig. 2). Neither procedure involved adjustable parameters and both yielded very similar outcomes (Supplementary Fig. 2). These results show that the motion of paired coding or regulatory elements can be tracked with relatively high accuracy using a combination of wild-type and mutant TET operator binding sites.

V H -D H J H trajectories revealed highly constrained motion

Individual trajectories of the spatial distances separating the labeled V H and D H J H regions revealed that, for the majority of cells (90%), V H -DJ H motion was highly constrained: the V H and D H J H segments that were initially in spatial proximity remained in proximity, whereas the segments that were initially spatially remote explored their immediate neighborhood while remaining remote (Fig. 3a and Supplementary Fig. 3). The corresponding probability distribution of V H -D H J H distances that was generated from these trajectories covered a broad range of distances (0.2–1.2 μm) suggesting that, across the population of cells, the locus adopts a wide spectrum of chromatin configurations (Fig. 3a, right panel). Notably, the distribution exhibited bimodality (p-value of 0.003 returned by Hartigan’s Dip Test indicated that bimodality was statistically significant) that persisted for at least 400 s, indicating that at least two dominant types of chromatin configurations were associated with Igh locus topology. Color-coding the temporal trajectories of V H -D H J H spatial distances according to their mean values revealed an intriguing “demixing” effect: the distances within the individual V H -D H J H pairs fluctuated around their respective mean values that remained nearly constant, together resulting in a “rainbow” pattern that persisted for at least an hour (Fig. 3a, left panel; Supplementary Fig. 3). These data indicate that the Igh locus adopts a wide spectrum of configurations that are sufficiently dynamic to allow local motion yet stable enough to provide the long-term confinement that either co-localizes V H and D H J H segments or isolates them from each other.

Fig. 3 V H -D H J H motion in B-cell progenitors is extremely confined. a The spatial distances separating V H and D H J H elements for the majority of cells fluctuated around their respective average values that remained nearly constant over time. Color-coding the distance trajectories according to their mean values reveals a “demixing” effect. Right panel shows the corresponding probability distribution of V H -D H J H distances. b The time-averaged radial MSD (colored lines) and the time-and-ensemble-averaged radial MSD (black line) for V H -D H J H motion. c Time-and-ensemble-averaged MSD of relative genomic motion for inter-chromosomal D H J H -D H J H pair (green) and intra-chromosomal V H -D H J H pair (red) before (pale lines) and after (bright lines) measurement-error correction. d Velocity autocorrelation functions for the V H -D H J H motion exhibit anti-correlations indicative of a push-back from the environment; the value at the dip approaches the theoretical limit (−0.5) of an extreme confinement. The collapse of the velocity autocorrelation function curves upon rescaling of the time axis (right panel) indicates self-similarity of motion. Source data are provided as a Source Data file. e Left panel shows the V H -D H J H trajectories that, unlike those in a, exhibited substantial changes in their mean values over time. Right panel shows the radial MSD curves (colored lines) for the individual cells that displayed abrupt changes in V H -D H J H motion and, for comparison, the time-and-ensemble-averaged radial MSD (black line) for V H -D H J H motion in the majority of cells. Scaling exponents (α) are indicated. Source data are provided as a Source Data File Full size image

The properties of V H -D H J H motion averaged across the trajectories were examined by computing the mean-squared displacements (MSD) and the velocity autocorrelation functions (Fig. 3b–d). The velocity autocorrelation function reveals the degree of correlation between the average velocities of a pair of segments at two instants separated by the time interval τ (the velocity itself is the average displacement over the time interval δ). MSD and velocity autocorrelation function were calculated using pairwise V H -D H J H and D H J H -D H J H distance trajectories, which eliminated the effect of nuclear motion. Time-averaged as well as time-and-ensemble-averaged MSD exhibited a subdiffusive scaling exponent (α < 1) both for intra-chromosomal V H -D H J H and inter-chromosomal D H J H -D H J H motion (Fig. 3b, c and Supplementary Fig. 4; Supplementary Table 1). Notably, V H -D H J H motion was found to be extremely subdiffusive, characterized by a scaling exponent α that decreased from 0.35 at short time scales to 0.2 at long time scales (Fig. 3c and Supplementary Table 1). The velocity autocorrelation functions exhibited negative correlations indicative of a push-back from the environment (Fig. 3d). For intra-chromosomal motion, the value of the velocity autocorrelation function at the dip approached the theoretical limit (−0.5) of an extreme confinement (Fig. 3d). The collapse of the velocity autocorrelation curves upon rescaling of the time axis indicated that the motion was self-similar, i.e., exhibited similar patterns at different spatial and temporal scales (Fig. 3d, right panel). Thus, the trajectories from the vast majority of imaged cells displayed substantial spatial confinement imposed on the V H -D H J H motion. We note, however, that a significant fraction (10%) of cells revealed trajectories that appeared less constrained and displayed abrupt changes in the scaling exponent ranging from 0.2 to 0.8 during the course of imaging (Fig. 3e). Taken together, these measurements indicate that, while in the majority of pro-B cells V H -D H J H motion was strongly subdiffusive and predominantly governed by a viscoelastic and spatially confining neighborhood, a subset of pro-B cells revealed V H -D H J H trajectories operating in a significantly less constrained environment.

Chromatin loops provide global confinement

To explore the mechanistic origin of the viscoelasticity and confinement that govern V H -D H J H motion, we modeled the chromatin fiber as a bead-spring polymer using molecular dynamics (MD) simulations (Fig. 4)48. Given the large number of molecular components and an extensive parameter space that could be incorporated in the simulations, we constrained the model by utilizing multiple independent sets of experimental data. As a first step, we sought to identify the minimal model that could reproduce the structural properties of the Igh locus, namely the plateau in the V H -D H J H spatial distances as a function of the genomic distance as observed in 3D-FISH49,50. As a second step, the resulting minimal model was refined such that it could also reproduce the dynamic properties of the locus, namely the near-constant average spatial distances separating the V H from the D H J H regions and the strongly subdiffusive scaling exponent associated with V H -D H J H motion.

Fig. 4 Molecular dynamics simulations capture Igh locus structure and dynamics. a The mean spatial separation as a function of the genomic distance between the D H J H and the markers that span the V H regions are indicated. Triangles represent the mean spatial separation between the marked regions and the D H J H elements as measured previously by 3D-FISH49. Note that the plotted average values were obtained for > 100 RAG-deficient pro-B cells for all shown data points49. The square and circle represent the mean spatial separation between the D H J H region and the V H 3 regions cultured in the absence (n = 121) or presence (n = 45) of STI-571, respectively. Colored lines show spatial separation as a function of genomic distance for the IgH locus modeled as an unstructured chromatin chain (blue line), a single loop configuration (red line) or a two-loop configuration (orange line). Right panel indicates the different configurations that were analyzed. b A snapshot extracted from MD simulations of the bead-spring polymer model with cross-linkable sites spanning the Igh locus. c Two representative trajectories of V H -D H J H spatial distances from simulations (red and blue lines) demonstrate how the insertion of crosslinks across the locus in the simulations reproduces the experimentally observed “demixing” of the trajectories. Source data is provided as a Source Data File Full size image

To capture the structural properties of the Igh locus, we simulated a hierarchy of worm-like chain polymer models reflecting chromatin configurations of increasing complexity: an unstructured chromatin chain, a single loop configuration, a two-loop configuration, and a chain enclosed in a confinement that mimicked a multiple-loop configuration (Fig. 4a and Supplementary Methods). As expected, the unstructured chain model failed to generate a plateau-like relationship between spatial and genomic V H -D H J H distances (Fig. 4a). A spatial confinement enclosing the chain was necessary to reproduce the plateau observed in 3D-FISH (Fig. 4a, solid black line)49,50. Notably, the average over a large number of explicit multiple-loop configurations in the absence of confinement also reproduced the plateau in the spatial vs. genomic distances (Fig. 4a, dotted black line). These results indicate that chromatin loops can act as a primary source of large-scale confinement to provide an equal playing field for the V H region repertoire independent of genomic separation.

Immunoglobulin heavy chain locus in a weak-gel state

While the model of the Igh locus organized as loops reproduced the structural properties of the locus, it yielded spatial distances that fluctuated considerably, resulting in intersecting trajectories (Fig. 4c, left panel). These trajectories were inconsistent with the “demixed” V H -D H J H trajectories observed in live-cell imaging, in which the spatial distances only exhibited local fluctuations (Fig. 3a and Supplementary Fig. 3). This discrepancy pointed to yet another layer of spatial constraint, which confined the motion locally. A plausible mechanism of the local confinement is cross-bridging of the chromatin fiber, analogous to the mechanism recently proposed for super-enhancers whereby crosslinks induce phase-separated condensates13. To examine a potential role for crosslinks as the source of local confinement, we introduced 5% cross-linkable sites that could undergo pairwise binding and unbinding with tunable kinetics (Fig. 4b and Supplementary Methods). Notably, gradually increasing the cross-link residence time resulted in a progressive suppression of the fluctuations in the spatial distances separating the paired genomic elements, ultimately recovering the effect of “demixing” of the trajectories (Fig. 4c).

A network of crosslinked polymer chains and a solution of free (un-crosslinked) chains represent two distinct physical phases: a gel phase (a solid phase) and a sol phase (a liquid phase), respectively51,52. Given that the ordered nature of the solid phase and the fluidity of the liquid phase can both be advantageous from a biological prospective, we asked: to what extent are the properties of Igh locus solid-like as opposed to liquid-like? In other words, how deep in the gel phase is the locus positioned on a phase diagram? The tuning of the bond lifetime of the simulated crosslinks from irreversible bonds (strong gel) to short-lived bonds (weak-gel) and further to the absence of crosslinking (sol) resulted in a systematic change in the slope (α) of the MSD (Fig. 5a). The bond lifetime on the order of 10 s was found to yield the best agreement between simulated and experimentally measured MSD. The short-lived nature of the crosslinks indicates that the state of the locus corresponds to a weak gel poised close to the boundary of gel and sol phases (Fig. 5a). These results suggest that a global confinement imposed by chromatin loops and a local confinement imposed by the cooperative action of short-lived crosslinks together constitute the mechanistic origin of the observed strongly subdiffusive V H -D H J H motion.

Fig. 5 A weak-gel model reproduces V H -D H J H first-encounter times. a Tuning the lifetime of crosslinks results in a systematic change in the slope (α) of the MSD. Short-lived bonds (lifetime ~10 s) yield the best agreement between MSD derived from experiment and simulations. b Representative trajectories of the spatial distance within individual V H -D H J H pairs in STI-571 treated cells (n = 121). Red circles indicate potential first encounters. c Representative trajectories of V H -D H J H spatial distances generated from the MD simulations of the Igh locus when modeled as a weak gel. d First encounter times as a function of mean spatial separation from experimental and simulated V H -D H J H trajectories (left panel), and the corresponding probability distributions of experimentally derived and simulated first-encounter times (right panel). Source data is provided as a Source Data File Full size image

V H -D H J H encounter times in a weak-gel state