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. 2014 Jul 17;158(2):339-352.
doi: 10.1016/j.cell.2014.05.036. Epub 2014 Jul 3.

3D trajectories adopted by coding and regulatory DNA elements: first-passage times for genomic interactions

Joseph S Lucas  1 , Yaojun Zhang  2 , Olga K Dudko  3 , Cornelis Murre  4
Affiliations

3D trajectories adopted by coding and regulatory DNA elements: first-passage times for genomic interactions

Joseph S Lucas et al. Cell. .

Abstract

During B lymphocyte development, immunoglobulin heavy-chain variable (VH), diversity (DH), and joining (JH) segments assemble to generate a diverse antigen receptor repertoire. Here, we have marked the distal VH and DH-JH-Eμ regions with Tet-operator binding sites and traced their 3D trajectories in pro-B cells transduced with a retrovirus encoding Tet-repressor-EGFP. We found that these elements displayed fractional Langevin motion (fLm) due to the viscoelastic hindrance from the surrounding network of proteins and chromatin fibers. Using fractional Langevin dynamics modeling, we found that, with high probability, DHJH elements reach a VH element within minutes. Spatial confinement emerged as the dominant parameter that determined the frequency of such encounters. We propose that the viscoelastic nature of the nuclear environment causes coding elements and regulatory elements to bounce back and forth in a spring-like fashion until specific genomic interactions are established and that spatial confinement of topological domains largely controls first-passage times for genomic interactions.

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Figures

Figure 1
Figure 1. Generation of Igh-TetO Labeled Mice
(A) Schematic of the Igh locus showing VH, DH, JH, and CH segments, the intronic enhancer (Eμ), and switch region repeats (Sμ). Dashed boxes show genomic regions that were replaced by arrays containing 240 copies of the Tet operator. Southern blot screening strategies are indicated using HindIII (H) or KpnI (K) restriction enzymes. Blue arrows show positions of genotyping primers. (B) Southern blot of embryonic stem cell clones positive for integration of the TetO arrays positioned adjacent to either the VH or DHJH region of the Igh locus. (C) PCR-based genotyping results of mice harboring TetO arrays on both (+/+), one (+/−), or neither (−/−) alleles of the Igh locus. See also Figure S1 and Table S3.
Figure 2
Figure 2. Characterization of Igh-TetO Labeled Mice
(A) Femoral bone marrow cells from wild type, DHJH-TetO, and VH-TetO mice stained with B cell markers CD19 and B220. Bottom panel shows CD19+B220+ cell numbers as a fraction of live cells. (B) Bone marrow cells stained for CD25 and C-kit. The lineage negative (CD11b, Gr1, Ter119) and B220+CD19+ population is shown. (C) DH-JH and VHJ558-DHJH rearrangements in B220+ cells isolated from wild type, DHJH-TetO, VH-TetO and Rag−/− mice analyzed by Southern blotting using probes corresponding to VH and DHJH gene segments See also Table S3.
Figure 3
Figure 3. Generation and Optimization of TetR-EGFP Expression
(A) Construction of TetR-EGFP expressing retroviral vectors. The NLS-TetR-EGFP coding sequence was cloned into three distinct retroviral vectors, MinV, PCS-Ret, and LMP. PGK or Gag promoters were used to drive EGFP expression in each of the vectors. Each construct was made using either a wild-type or mutant Kozak sequence. In the MinV and LMP vectors, the NLS-TetR-EGFP was inserted 3′ of an internal ribosomal entry site (IRES). (B) DHJH-TetO B-lineage cells were infected with NLS-TetR-EGFP expressing retrovirus and expression levels were monitored using flow cytometry. Green bars show approximate expression levels leading to high signal to noise ratios of TetO-EGFP mediated fluorescence. (C) DHJH-TetO B-lineage cells infected with NLS-TetR-EGFP in LMP with a mutant Kozak sequence and visualized by fluorescence microscopy. The dots in each cell show both Igh alleles. See also Movies S1 and S2.
Figure 4
Figure 4. A Viscoelastic Environment Dominates Igh DHJH and VH motion in Pro-B Cells
(A) Model depicting fractional Langevin motion across the Igh locus. The Igh locus is organized as bundles of loops, with CTCF (purple) positioning the VH segments (red) in orbit around the DHJH region (blue). Enlarged region depicts the viscoelastic properties of the chromatin fiber caused by interactions with neighboring networks of nucleic acids and proteins as well as restoring forces within the fiber itself (shown as springs). (B) Time-averaged radial MSD plotted as a function of time lag (τ) for the DHJH region in live B220+ B-lineage cells isolated from TetO mice. Number of cells (N) analyzed is indicated. Cells were imaged once every 2 seconds for 400 seconds (top panel) or once every 40 seconds for 4000 seconds (bottom panel). (C) Ensemble- and time-averaged radial MSD plotted as a function of time lag (τ) for the VH and DHJH regions in live B220+ B-lineage cells as well as formaldehyde fixed pro-B cells. Radial MSD is shown for τ up to one half of total imaging time. Shaded areas represent the standard error of the mean. Dashed lines indicate a sub-diffusive scaling exponent (α) of 0.5. (D) Velocity autocorrelation analysis of DHJH regions in live B220+ B-lineage cells (left panels). Average velocity was calculated over discretization intervals (δ) ranging from 2 to 200 seconds in 2 seconds intervals (top panel) or 40–2000 seconds in 40 seconds intervals (bottom panel). Velocity autocorrelation curves for different values of δ, plotted against a rescaled time lag (τ/δ) (right panels). The color scheme represents the values of δ from small (blue) to large (red). See also Figures S2 and S3 and Tables S1 and S2.
Figure 5
Figure 5. 3D-Trajectories Adopted by the Immunoglobulin Heavy Chain Locus Motion in Wild-Type and Rag-deficient pro-B Cells
(A) Ensemble- and time-averaged radial MSD plotted as a function of time lag (τ) for the DHJH regions in live B220+ B-lineage cells derived from wild type and Rag−/− mice. Cells were imaged once every 2 seconds for 400 seconds (top panel) or once every 40 seconds for 4000 seconds (bottom panel). Radial MSD is shown for τ up to one half of total imaging time. Shaded areas represent the standard error of the mean. (B) Velocity autocorrelation analysis of DHJH regions in wild type and Rag−/− cells. Velocity was calculated over time intervals (δ) ranging from 2 to 40 seconds in 2 seconds steps (top panel) or 40–800 seconds in 40 seconds steps (bottom panel).
Figure 6
Figure 6. Modeling Anomalous Diffusion of VH and DHJH Elements as Fractional Langevin Motion
(A) VH and DHJH segments confined in a sphere of radius R and subject to fractional Langevin motion using experimentally obtained values of anomalous diffusion coefficient (D) and subdiffusive exponent (α) as physical parameters. (B) Radial MSD obtained from simulated (D=0.0024 μm2/s0.5 and R=1 μm) and experimental measurements. Black symbols are calculated from simulated trajectories. Gray symbols are DHJH MSD calculated from measurements in wild-type pro-B cells. (C) Velocity autocorrelation functions generated from simulations with velocity computed for different values of the discretization interval (δ) exhibit a collapse on a master curve upon rescaling of the time lag τ by δ, as do their experimentally determined counterparts (Fig. 4D). (D) The steady-state distribution of distances between a DHJH and a VH segment generated from the simulation (D=0.0024 μm2/s0.5 and R=0.5 μm) agrees well with the experimental distribution of spatial distances previously measured in pro-B cells by 3D-FISH (Jhunjhunwala et al. 2008). See also Movies S3 and S4.
Figure 7
Figure 7. First-Passage Time Distributions for VH and DHJH Segments Undergoing fractional Langevin Motion
(A) Simulating the behavior of a single pair of VH and DHJH segments in a spatial confinement of radius 1.0 μm and with initial positions randomly chosen from the distribution shown in Figure 6D generated after the system is given enough time to reach the steady state. Motion of VH and DHJH segments as parts of the polymer chain and constrained by anchors is modeled as fractional Langevin motion in a confined sphere of radius R. The distribution of first passage times, the times for the DHJH segment to come within 30 nm of the VH segment, is shown. Red arrow indicates mean first passage time (MFPT). (B) First passage time distributions for simulated motion of 100 VH and one DHJH segment in a confinement of radius 1.0 μm. (C) Left panels show 3D-FISH studies revealing the organization and locus contraction of the Igh locus in pre-pro-B and pro-B cells. In pre-pro-B cells, VH and DHJH segments are physically separated into distinct topological domains. Upon differentiating into committed pro-B cells the proximal and distal VH regions merge. Right panel indicates the entire Igh locus labeled by overlapping fluorescently labeled BAC probes. Note distinct topological domains in pre-pro-B and pro-B cells (adapted from Jhunjhunwala et al., 2008). (D) Simulation as performed in (B) but with VH segments distributed on a sphere with the DHJH segment in the center. (E, F) Simulations as performed in (A) and (B) but with a confinement of radius 0.5 μm.
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