The IGH locus relocalizes to a "recombination compartment" in the perinucleolar region of differentiating B-lymphocytes

Abstract

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.

Keywords: Chromosome Section; chromatin; differentiation; immunoglobulin genes; recombination.

Figure 1. Radial positions of productive and non-productive IGH alleles in human B-lymphocytes

A. A productive IGH allele can be discriminated by FISH in B-cells after V(D)J recombination. Top, a schematic representation of human chromosome 14 and two different IGH loci: an IGH allele with a germline structure of IgH locus contains two genomic fragments stained by BACs RP11-346I20 (green) and RP11-259B19 (red); the IGH allele which underwent V(D)J recombination and expresses functional antibodies hybridizes only with RP11-346I20 (green) which is localized upstream from constant segment. Bottom, a typical image of FISH and immunostaining is shown. Scale bar = 5 μm. B. Radial positions of productive and non-productive IGH-alleles measured in the nuclei of Day₀ cells (n = 75), Day₁ cells (n = 102), Day₄ cells (n = 113), Day_5/6 cells (n = 81), and Plasmocytes (n = 36). Black line shows median, 0 - nuclear center, 1 - nuclear periphery. P-values were calculated using Tukey's multiple comparison test (ns, non-significant; ** <0.01, ****<0.0001); adjusted p-values for all pairs of means are presented in Table S1. Distances were measured as described in Materials and methods. Scatter plots represent the distribution of FISH signals in the nuclear space. The nuclei were divided into concentric spheres with the equal volume.

Figure 2. IGH alleles do not colocalize with heterochromatin clusters

A. Pearson's coefficients for colocalization of IGH H3K9me3 and IGH in B-cells from Day₀, Day₁, Day₄ and Day_5/6 show the absence of colocalization. B. Top, Immuno-FISH with the BAC probe RP11-346I20 (green), and either DAPI (blue, B) or anti-H3K9me3 (red, C); middle, FISH signals of the IGH loci; Bottom, 2D representation of Pearson's coefficients for colocalization in Day₀, Day₁, Day₄ and Day_5/6 B-cells. Scale bar = 10 μm.

Figure 3. The ectopically expressed AID-GFP forms a unique perinucleolar focus associated with IGH at the periphery of nucleoli

A., B., BL2 cells expressing AID-GFP fixed, hybridized and stained 48h after stimulation. Typical images of immuno-FISH staining showing the colocalization of TOPRO3, AID-GFP (green), IGH (red, B) or nucleolin (C23; red, C). Scale bar = 5 μm.

Figure 4. Localization of productive and non-productive IGH-alleles relative to nucleolus at different stages of B-cell differentiation

A. Mean distances between nucleolus and the IGH alleles measured in the nuclei of Day₀ cells (n = 76), Day₁ cells (n = 102), Day₄ cells (n = 113), Day_5/6 cells (n = 81), and Plasmocytes (n = 37). Error bars represent 95% CI. P-values were calculated using Tukey's multiple comparison test (ns, non-significant; ** <0.01); adjusted p-values for all pairs of means are presented in Table S2. B. Percentage of productive and non-productive IGH-alleles localized at nucleolus (left), within a distance of 0.1 µm to nucleolus (middle) or within a distance of 0.25 µm to nucleolus (right), as calculated for the cells analyzed in (A).

Figure 5. IGH colocalizes with the nucleolus-proximal γH2AX foci in Day_5/6 B-cells

A. AID expression in differentiating B-lymphocytes measured by qPCR relative to GAPDH expression. B. Staining of γH2AX foci in differentiating B-lymphocytes. IGH (green), γH2AX (red) and nucleoli (B23, blue) were simultaneously revealed by immuno-FISH. Scale bar = 5µm. C. Percentage of cells containing the IGH locus colocalizing with γH2AX or with both γH2AX and nucleolus. Columns represent means with 95% CI (error bars) for several independent calculations (Day₀, n = 10; Day₄, n = 8; Day_5/6, n = 10). P-values were calculated using Tukey's multiple comparison test (ns, non-significant; *** <0.001; **** <0.0001).