The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions

Abstract

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

Figure 1. Immunoglobulin Heavy-Chain Locus Spatial Distances and Spatial Distributions as a Function of Genomic Separation in Pre-Pro-B and Pro-B cells

(A) Genomic organization of the Igh locus. The anchor and the genomic markers used are indicated. (B) Frequency plots showing the distribution of spatial distances between the probes and the anchor (RP23-201H14). Cumulative frequency distributions are indicated for both pre-pro-B and pro-B cells. (C) Average spatial distances were plotted as a function of genomic separation. Distal and proximal variable regions as well as diversity, joining and constant region segments are shown. Bars indicate standard error. The dotted lines only indicate connectivity. The arrow indicates the position of the intronic enhancer.

Figure 2. Immunoglobulin Heavy-Chain Locus Topology in Pro-B Cells Brings Distal V_H Regions and D_HJ_H and Enhancer Elements in Close Spatial Proximity

(A) Genomic organization of the Igh locus. The anchor and the genomic markers used are indicated. (B) Probe h4 contains the J_H segments, the intronic enhancer and C_μ elements. (C) Frequency plots showing the distribution of spatial distances for each genomic marker from the Igh D_H-J_H cluster. Cumulative frequency distributions are indicated for both pre-pro-B and pro-B cells. (D) Average spatial distances were plotted as a function of genomic separation for each of the probes. Distal and proximal variable regions as well as diversity, joining and constant region segments are shown. Bars indicate standard error. The dotted lines only indicate connectivity. The arrow indicates the position of the intronic enhancer.

Figure 3. Immunoglobulin Heavy-Chain Topology and Comparison to the Self-Avoiding Random Walk and Worm-like Chain

(A–D) Scaled distributions of spatial distances were plotted for comparison with the self-avoiding walk. A scaling component of v = 0.6 was used. Graphs are shown for both cell types, and with either BAC RP23-201H14 or h4 probes as anchors. (E) Comparing Igh topology to a worm-like chain with a range of chromatin densities (100–10,000 bp/nm) but with a constant persistence length (200 nm). A “least square optimization” analysis is also shown (dotted line). (F) Spatial distances were plotted as a function of genomic separation and compared with the Porod-Kratky chain with a range in persistence length of 50–200 nm and a constant chromatin density of 130 bp/nm.

Figure 4. Comparison and Evaluation of Spatial Distances between Genomic Markers in the Immunoglobulin Heavy-Chain Locus by Computer Simulations

(A) Volume rendered images of simulated Random-Walk/Giant-Loop and Multi-Loop-Subcompartment Models. As a starting conformation with the form and size of a metaphase chromosome (top), rosettes were stacked (α). From such a starting configuration, interphase chromosomes in thermodynamic equilibrium, were decondensed by Monte-Carlo and relaxing Brownian Dynamics steps. A volume rendered image of the simulated Random-Walk/Giant-Loop model containing large loops (5 Mbp) is shown (left; β). Note that the large loops do not form distinct structures but intermingle freely (left; β). In contrast, in a volume rendered image of the simulated Multi-Loop-Subcompartment Model, containing 126 kbp sized loops and linkers, the rosettes form distinct chromatin territories in which the loops do not intermingle freely (middle; γ). Also is indicated an image of the simulated RW/GL model containing 126 kbp loops and 63 kbp linkers (right; δ). Note that the small loops do not intermingle freely. Distinct chromatin territories cannot be detected (Knoch, 2003). (B) Strategy for position-dependent and position-independent virtual spatial distance measurements. For position-dependent virtual distance measurements, the anchor was placed close to the base of the loop (marker 1). The virtual spatial distances were measured from the anchor to other makers in the rosette (1–7) and to a linker (8–10). For position-independent measurements a set of markers separated by the same genomic distance were randomly positioned (x,y,z). (C) Comparison between simulated position-dependent (dotted lines) and position-independent (solid lines) spatial distances. The curves (A–D) indicate simulated MLS models with 126 kbp loops and different linker sizes. RW/GL is shown for comparison (a). Position-dependent distances (dotted lines) show a stepwise increase in the region where a linker is connecting two chromatin sub-compartments, while position-independent distances (solid lines) do not show the stepwise increase in spatial distances as a function of genomic separation. (D) Random-Walk Giant Loop and Multi-Loop-Subcompartment Models. α indicates the RW/GL model in which large loops are attached to a non-DNA backbone. β shows the simulated model containing a chromatin linker between loops. MLS model is shown containing 126 kbp loops and linkers with individual rosettes spanning 1–2 Mbp (Knoch, 2003). (E) The simulated spatial distances as a function of genomic separation are shown for a fixed loop structure. The simulated loop size was 126 kbp. Two virtual genomic markers were chosen that were separated by 252 kbp. The heat map indicates the frequency distribution of simulated spatial distances. (F and G) Comparison between experimental data and computer simulated data obtained from spatial distance measurements in the Igh locus as a function of genomic separation (5.2 kbp steps). Nomenclature is loop size (kbp)-linker size (kbp)-topology. Experimental spatial distance measurements (μm) were plotted as a function of genomic separation (Mbp) for pre-pro-B cells (blue dots and green circles) and pro-B cells (red squares and pink triangles).

Figure 5. 3D Topology of the Immunoglobulin Heavy-Chain Locus

The 3D topology of the Igh locus in pre-pro-B and pro-B cells was resolved using trilateration. The relative positions of 12 genomic markers spanning the entire immunoglobulin heavy-chain locus were computed. Two different views are shown for both cell types. (A) 3D Topology of the Igh locus in pre-pro-B cells. (B) 3D Topology of the Igh locus in pro-B cells. Grey objects indicate C_H regions and the 3′ flanking region of the Igh locus. Blue objects indicate proximal V_H regions. Green objects indicate distal V_H regions. Red line indicates the linker connecting the proximal V_H and J_H regions. Linkers are indicated only to show connectivity.

Figure 6. Visualization of the Entire Immunoglobulin Heavy-Chain Locus

(A) Igh locus was fluorescently labeled using BACs that span the entire locus. (B) 3D FISH in nuclei derived from pre-pro-B and pro-B cells using fluorescently labeled BACs that span the entire Igh locus. Digitally magnified pictures of the Igh locus are shown. BACs (shown in red) were directly labeled with dUTP conjugated to Alexa 568. Nuclei were visualized by DAPI staining. (C) Fractions of the number of compartments visualized using overlapping set of BAC probes in pre-pro-B and pro-B cells are indicated.

Figure 7. Probabilities of Long-Range Interactions in Pre-pro-B and Pro-B Cells Compared to Those Predicted by a 3D Random Walk

Cumulative frequencies were obtained by accumulating the frequency values corresponding to the spatial distances in intervals of 100 nm using the D_HJ_H elements (probe h4) as an anchor. Cumulative frequency distributions for the random walk were determined for different persistence lengths (see Experimental Procedures). The cumulative frequency distribution for the spatial distance between h4 and h5 is similar for pre-pro-B cells, pro-B cells and for a random walk with a persistence length of 50 nm and chromatin density of 130 bp/nm.