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. 2013 Feb 15;190(4):1819-26.
doi: 10.4049/jimmunol.1203127. Epub 2013 Jan 7.

Vκ gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Vκ-Jκ intervening region

Yougui Xiang  1 , Sung-Kyun Park, William T Garrard
Affiliations

Vκ gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Vκ-Jκ intervening region

Yougui Xiang et al. J Immunol. .

Abstract

The processes of Ig gene locus contraction and looping during V(D)J-recombination are essential for creating a diverse Ab repertoire. However, no cis-acting sequence that plays a major role in specifying locus contraction has been uncovered within the Igκ gene locus. In this article, we demonstrate that a 650-bp sequence corresponding to DNase I hypersensitive sites HS1-2 within the mouse Igκ gene V-J intervening region binds CCCTC-binding factor and specifies locus contraction and long-range Vκ gene usage spanning 3.2 Mb in pre-B cells. We call this novel element Cer (for "contracting element for recombination"). Targeted deletion of Cer caused markedly increased proximal and greatly diminished upstream Vκ gene usage, higher allele usage, more splenic Igκ(+) B cells, and nonlineage-specific Igκ rearrangement in T cells. Relative to wild-type mice, Cer-deletion mice exhibited similar levels of Vκ gene germline transcription and H3K4me3 epigenetic marks but displayed a dramatic decrease in locus contraction in pre-B cells. Thus, our studies demonstrate that DNase I hypersensitive sites HS1-2 within the Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire.

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Figures

FIGURE 1
FIGURE 1
Map of the mouse Igκ gene locus, highlighting features of the Vκ–Jκ intervening region. A, Schematic diagram of the Igκ gene locus, with exons indicated as closed rectangles, and cis-acting sequences indicated as open rectangles, which include Sis, 5′ and 3′ germline promoters (Gp), and downstream enhancers (E). T indicates the termination region of transcription. The arrows at the locus boundaries indicate the directions of transcription of flanking housekeeping genes. B, Schematic diagram of the Vκ–Jκ intervening region indicating the location of DNase I hypersensitive sites 1 to 6 (vertical arrows). The bracketed horizontal dashed lines indicate the positions of deletion mutants. The horizontal arrows indicate the directions of transcription from a Vκ21 gene and the 5′ germline promoter (5′Gp). C, In silico predicted CTCF binding sites within HS1-2 and HS3-6 (Sis). The scores for these predicted CTCF binding sites were larger than 10. Usually a sequence with a score >3.0 is a suggestive match for a CTCF binding site (42). Base mismatches between the site pairs are depicted in grey. D, CTCF enrichment in HS1-2 and HS3-6 in pre-B cells from Rag1−/−μ+ mice as assayed by ChIP. Vκ9-132 is known to possess CTCF binding positive sites and served as a positive control, whereas Vκ2(−) lacks such sites and served as a negative control (43). Data are the means ± SD of 3 independent experiments. E, HS1-2 deletion eliminates localized CTCF binding in pre-B cells. Real-time PCR ChIP assays of CTCF occupancy for WT and HS1-2−/− samples. Map of the HS1-2 element and upstream PCR primers used in the ChIP assay common to alleles possessing or lacking HS1-2 (upper, small arrows). Results are the means ± SD of 3 independent experiments.
FIGURE 2
FIGURE 2
HS1-2−/− mice exhibit increased Igκ+ B cells. A, FACS analysis of cell surface Ig expression in WT and HS1-2−/− mice splenic cells. Single cell suspensions from spleen were stained with anti-B220 and anti-Igκ antibodies (upper); or anti-B220 and anti-Igλ antibodies (lower). Only cells residing in the live lymphocyte gate were analyzed. Percentages of cells residing in various windows are shown in the figure sub-panels. Data are representative of independent FACS analyses from at least 10 mice of each genotype. For B220+Igκ+ cells: HS1-2−/− vs WT, p=0.004; (Student’s t-test). B, Igκ vs Igλ usage and IgL isotype exclusion in WT and HS1-2−/− splenic cells were analyzed by FACS. C, FACS analysis of the effect of HS1-2 deletion on mCκ allele usage and Igκ allelic exclusion.
FIGURE 3
FIGURE 3
HS1-2−/− mice exhibit dramatically altered Vκ gene usage. A, The Vκ-Jκ1 rearrangement products of pre-B cells from WT, HS1-2−/− and HS3-6−/−(Sis−/−) mice were amplified from genomic DNA by PCR and cloned into the pGEM-T vector. More than 100 independently determined Vκ gene sequences from each group were identified by IgBlast. Their percent usage relative to the total Vκ gene usage as 100% are presented in 0.1 Mb interval distances from the Jκ-region along the Igκ gene locus. B and C, Analysis of relative Vκ gene usage by real-time PCR assays. Vκ gene specific primers and a primer downstream of Jκ1 were used to assay for specific Vκ-Jκ1 rearrangements in genomic DNA samples. The % usage of Vκ genes was compared with those of WT mice in which the % usage was set as 100% (depicted as dashed lines). Data are means ± SD of 3 independent experiments. The distances of these Vκ genes from the Jκ1-region are: Vκ2-139, 3,085 kb; Vκ9-132, 2,854 kb; Vκ9-122, 2,740 kb; Vκ19-15, 311 kb; Vκ21-7, 111 kb; Vκ21-1, 18 kb (8). B, Usage of different Vκ genes in pre-B cells from HS1-2−/− mice. C, Usage of different Vκ genes in splenic B cells from HS1-2−/− mice.
FIGURE 4
FIGURE 4
Analysis of Jκ-region usage, and the developmental timing and tissue-specificity of Vκ–Jκ rearrangement. A, Schematic diagrams (not to scale) of the PCR assays used for determining Jκ-region usage or N and P nucleotide insertions. The top map shows the positions of a degenerate VκD gene 5′ primer, and J1r and Mar35 3′ primers along an Igκ gene germline locus. Also shown is the position of a probe (solid bar) used in Southern blotting. Below are shown the four possible recombination products resulting from Vκ joining to the different Jκ–regions. B, Jκ-region usage analysis. The Vκ-Jκn rearrangement products of pre-B cell samples were PCR amplified using VκD and MAR35 primers. Reaction products were separated by electrophoreses on agarose gels, and the intensities of Vκ-Jκ1 to Vκ-Jκ5 bands were visualized by PhosphorImaging of Southern blots. The PCR amplifications of c-myb are shown at the bottom, which were used as loading controls for the amount of genomic DNA template in the PCR reactions. C, The relative usage of the indicated Jκ-regions determined by quantitation of Phosphorimages are shown as ratios for pre-B cell samples from HS1-2−/− mice compared with those of WT in which Jκ–region usage was set as 1. D, Vκ-Jκ rearrangement products of thymus double positive T (DPT) cells from the indicated genetic lines of mice were PCR amplified and the intensities of Vκ-Jκ1 to Vκ-Jκ5 bands were visualized by PhosphorImager analysis of Southern blots. E, Analysis of N and P nucleotides in Vκ-Jκ1 junction regions. The Vκ-Jκ1 rearrangement products were PCR-amplified using VκD and J1r primers from genomic DNA of pre-B cell samples from WT and HS1-2−/− mice and cloned into the pGEM-T vector. N and P nucleotide in more than 100 sequenced samples from each genetic mouse line were analyzed.
FIGURE 5
FIGURE 5
Levels of Vκ gene germline transcription and H3K4me3 modification in pre-B cells. A, Real-time PCR assays were used to measure Igκ gene germline transcripts arising from the 5′ promoter (5′GL) and from the indicated Vκ genes in pre-B cell samples from WT and HS1-2−/− mice. Data are means ± SD of 3 independent experiments. B, Real time PCR ChIP assays of H3K4me3 levels in Vκ gene RSS regions in pre-B cells of WT and HS1-2−/− mice. Data are presented as means ± SD (n=3). The distances of these Vκ genes from the Jκ1-region are given in Fig. 3.
FIGURE 6
FIGURE 6
HS1-2−/− pre-B cells exhibit reduced Igκ gene locus contraction. A, Map of the Igκ locus indicating the positions of color-coded BAC probes (A: RP23-101G13, red; B: RP23-26A6, blue; and C: RP24-387E13, green). The center-to-center distances between these probes in naked DNA are: A-B, 1,128 kb; B-C, 1,780 kb; and A-C, 2,908 kb. B, The left panels show representative 3D DNA FISH confocal optical sections from WT and HS1-2−/− mice pre-B cell nuclei, outlined by white dashed lines, as identified by DAPI DNA staining (white). The center panels represent enlargements of the corresponding hybridization images. The right panels depict schematic interpretations of the looping patterns of the loci based on the probe hybridization patterns. C, Dot plots representing measured center-to-center distances between the indicated hybridizing signals of the Igκ locus BAC probes. Data from 263 WT and 222 HS1-2−/− pre-B cell alleles were accumulated from measurements in several independent experiments. Mean distances are indicated by the horizontal lines, which are significantly different between WT and HS1-2−/− samples (Student’s t-test, A-B, p=0.004; B-C, p=2.6e−5; A-C, p=1.8e−17).
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