Figure 1.

Different transcribing Ig genes of plasma cells exhibit a pronounced colocalization in transcription factories, often near the nuclear periphery. (A) Representative four-color 3D RNA immuno-FISH results of the same 0.3-μm optical section of a plasma cell nucleus, outlined by white dashed lines. (Green) Igκi; (red) IgHi; (yellow) IgJi; (white) α-Pol II. As expected, ≥89% of Ig gene transcription was associated with transcription factories (Supplemental Fig. 1A), which averaged 155 ± 17 in number per plasma cell nucleus (Supplemental Fig. 1B). (B) Reconstructed 3D image of the same plasma cell nucleus shown in A, with the same color coding. (C) The percentages of cells that possess the indicated primary transcript localized <0.5 μm from a different Ig gene’s primary transcript are depicted in the overlapping images as quantified from 3D RNA FISH results for the 342 plasma cells examined that transcribed at least one allele of each of the Igκ, IgH, and IgJ genes. Data are from 470 cells analyzed in three independent experiments (see Supplemental Table 1 for primary data). The sums of the percentages in any one-gene circle total >100% because the same cells sometimes transcribed two different pairs of colocalized Ig genes (asterisks). (D–F) Bar graphs of 3D RNA FISH results from 607 pro-B cells, 618 pre-B cells, 608 imm-B cells, 625 B cells, 614 B + LPS cells, and 614 plasma cells analyzed from three independent experiments (see also Supplemental Tables 2–4 for primary data and P-values from χ² statistical tests). (D) The total number of transcribing alleles of Igκ, IgH, and IgJ genes per plasma cell and the percentage of cells for each case (data are represented as means ± SD). (E) Distributions of distances between transcribing homologous and/or heterologous allele gene pairs in plasma and other B cells. For homologous allele pairing, 374, 362, and 169 plasma cells that exhibited biallelic transcription for Igκ, IgH, and IgJ, respectively, were analyzed from three independent experiments. (F) Transcribed allele separation distances from the nuclear periphery in plasma and other B cells with and/or without consideration of allele pairing. For the distribution of distances with consideration of allele pairing, 307 plasma cells that had one colocalized Igκ•IgH allele pair were analyzed from three independent experiments.