Our previous studies have shown that DNase I hypersensitive sites 1 and 2 (HS1-2) and HS3-6 within the mouse Vκ–Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire. In this article, we demonstrate that a 6.3-kb deletion encompassing HS1-6 altogether not only leads to the predictable sums of these phenotypes, but also results in a novel hyperelevation of transcription of proximal Vκ genes, in both pre-B and splenic B cells. These findings reveal previously unrecognized additional functions for cis-elements within the Vκ–Jκ intervening region, namely, prevention of the production of massive levels of noncoding RNA species by silencing transcription of germline proximal Vκ genes in both developing and mature B cells.
This article is featured in In This Issue, p.3179
During B cell development, the Ig genes exhibit regulated V(D)J joining mediated by RAG-encoded recombinases and nonhomologous end-joining proteins, with the IgH gene locus rearranging first in pro-B cells followed by the IgL genes in pre-B cells (1). The Ig V gene Ab repertoire is generated not only by V(D)J joining, but also by receptor editing, somatic hypermutation, transcription levels of rearranged genes, and differential mRNA stabilities (2–5). For the immune system to efficiently recognize a broad spectrum of invading pathogens, diversity in the repertoire is essential (6, 7). Furthermore, misregulated or incorrect repertoire specification can trigger autoimmunity (8–10). Hence understanding the molecular mechanisms that specify repertoire may permit in the future novel ways to facilitate the production or maintenance of specific beneficial Abs, or the inhibition of the production or maintenance of detrimental species.
In the mouse, ∼95% of IgL chain species are contributed by the Igκ locus, which is the largest multigene family thus far identified, spanning 3.2 Mb on mouse chromosome 6 (11). It consists of 100 functional Vκ gene exons (12), 4 functional Jκ region exons, and a single Cκ exon. The Vκ gene repertoire resulting from this locus exhibits substantial diversity as assayed by a variety of techniques (4, 12–16). If Igκ gene V-J joining is productively unsuccessful because of out-of-reading frame recombination junctions, then the Igλ locus becomes activated for rearrangement and expression, which accounts for production of only 5% of the total IgL chains (17).
Vκ–Jκ recombination results in transcriptional activation because it positions a Vκ gene carrying its own promoter into a chromatin domain containing three powerful downstream enhancers: an intronic enhancer within the transcription unit and two enhancers downstream of the transcription termination region, termed E3′ and Ed (18–21). It is interesting to note, however, that before gene rearrangement, different mouse Vκ genes reside in either forward or reverse transcriptional orientations with respect to the Jκ-Cκ region (Fig. 1A, 1B). Rearrangement of forward orientation Vκ genes results in deletion of the 20-kb sequence in the Vκ–Jκ intervening region (Fig. 1A), which possesses six DNase I hypersensitive sites (HS1-6) (21). These sites include the cis-elements Cer (HS1-2) and Sis (HS3-6), which individually specify locus contraction and recombination silencer activities, respectively (15, 16, 22). By contrast, rearrangement of reverse orientation Vκ genes results in repositioning HS1-6 upstream in the locus by inversion (Fig. 1B). The closest Vκ gene in the reverse orientation resides 265 kb from Jκ1, whereas the farthest corresponding Vκ gene resides 2,780 kb from Jκ1 (11). Primary rearrangement events preferentially use Jκ1 (23, 24), reserving the downstream Jκ regions for receptor editing upon repeated recombination (e.g., Fig. 1B, lower) (25).
In previous studies, we have separately deleted HS1-2 and HS3-6 from the locus and elucidated the effects of these deletions on Igκ gene dynamics (15, 16, 22). In this investigation, we have created a major deletion of HS1-6 altogether in the mouse germline (Fig. 1C, 1D, and Supplemental Fig. 1), thus allowing us to determine whether a new and potentially unpredictable phenotype may result because of functional redundancies between Cer and Sis elements. Indeed, as a consequence of this deletion, we observed not only predictable alterations in the Vκ gene repertoire and reduced locus contraction in pre-B cells, but for the first time, to our knowledge, hyperelevated transcription of unrearranged proximal Vκ genes, both in pre-B and splenic B cells. Because of the efficiency of recombination, ∼40% of the B cells with productively rearranged Igκ genes will possess unrearranged allelic partners (26). These germline loci will still possess HS1-6 residing in between proximal Vκ genes and the downstream enhancers. Hence our results reveal that the function of the Vκ–Jκ intervening region is not only to specify repertoire in pre-B cells, but also to prevent the massive production of noncoding RNAs in Ab-producing B cells by silencing transcription of germline proximal Vκ genes.
Materials and Methods
Mice possessing a 6.3-kb deletion of HS1-6 in the endogenous Igκ locus were generated by standard embryonic stem (ES) cell targeting technology; germline transmissible mice were bred with Cre recombinase expressing MORE (27) mice to obtain HS1-6 and neor deletion mice (Fig. 1C, 1D, and Supplemental Fig. 1). Mice bearing a human Cκ knocked-in gene were kindly provided by Michel C. Nussenzweig of The Rockefeller University (2). All mice were used in accordance with protocols approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee. Additional details are provided in the supplemental data.
Flow cytometry and cell fractionation
Single-cell suspensions were prepared from spleen, bone marrow, and thymus of 6- to 14-wk-old mice. Single-cell suspensions were stained with Abs and analyzed using FACSCalibur with CellQuest software (BD Biosciences, San Diego, CA) or FlowJo software (Tree Star, Ashland, OR) (15). B220+CD43−IgM− small pre-B cells and CD4+CD8+ double-positive T (DPT) cells were sorted by a MoFlo flow cytometer. Splenic B cells were purified using B cell isolation kits with MACS cell separation columns (Miltenyi Biotec, San Diego, CA). Generally, we pooled bone marrow or splenic cells from two to three animals of the same genetic background for cell fractionation. Abs used are as follows: anti–mouse Igκ-PE (BD Biosciences); anti–mouse Igλ1,2,3-FITC (BD Biosciences); anti–human Igκ-FITC (Southern Biotech, Birmingham, AL); anti–B220-PerCP-Cy5.5, anti–IgM-allophycocyanin, anti–CD43-PE, anti–B220-FITC, anti–CD21-FITC, anti–CD23-allophycocyanin, anti–CD4-FITC, anti–CD8a-PE, anti–B220-biotin (all from BD Biosciences); and streptavidin-allophycocyanin (Southern Biotech).
Analysis of Igκ gene repertoire and Igκ V-J rearrangement
These assays were performed as previously described (15, 28). For analysis of Igκ gene repertoire of Vκ–Jκ1 rearrangement, genomic DNA was purified from sorted pre-B cell or DPT cell populations. The VκD primer and a primer in the Jκ1 intron were used to amplify Vκ–Jκ1 rearrangements; resulting PCR products were gel purified and subcloned into the pGEM-T vector (Promega, San Luis Obispo, CA; primer sequences are listed in Supplemental Table I). For analysis of Igκ gene repertoire of Vκ–Jκ5 rearrangement, the VκD primer and MAR35 primer in the Jκ5 downstream were used to amplify Vκ–Jκn rearrangements; resulting Vκ–Jκ5 PCR products were gel purified and subcloned into the pGEM-T vector. Determined sequences of Vκ genes in each clone were identified by the IgBlast program (National Center for Biotechnology Information, Bethesda, MD). For Igκ V-J rearrangement analysis, a quantitative PCR assay was performed as described previously (28). In brief, the Igκ V-J rearrangement products were PCR amplified by using the Platinum Taq high-fidelity DNA polymerase (Invitrogen, Carlsbad, CA) with a degenerate Vκ primer (VκD) and a MAR35 primer. PCR products were resolved in agarose gels and transferred to Zeta-probe GT genomic membrane (Bio-Rad Laboratories, Richmond, CA). The membranes were hybridized with a [α-32P] dCTP-labeled probe within the Jκ to intronic matrix association region plus enhancer region (28). Membranes were exposed to PhosphorImaging screens, and images were analyzed using ImageQuant software (Molecular Dynamics, Pittsburgh, PA).
Real-time PCR for Vκ–Jκ1 rearrangement, Igκ gene germline retention, and Igκ germline transcription
For real-time PCR analysis of Vκ–Jκ1 rearrangements or individual Vκ–Jκ1 rearrangements, the VκD primer or the forward primers specific to different Vκ exons and a reverse primer complementary to the Jκ1 intron region were used. Different Vκ–Jκ1 rearrangements were determined quantitatively by using the SYBR Green PCR master mix (Bio-Rad) in the 7300 real-time PCR system (Invitrogen) or the CFX connect real-time PCR detection system (Bio-Rad). PCR was performed based on manufacturer’s protocols, and each PCR assay was carried out in duplicate or triplicate. Relative rearrangements were calculated using the ΔΔ cycle threshold method according to the manufacturer’s instructions and normalized to a β-actin genomic region. To assay for germline retention of Igκ alleles, we used the forward and reverse primers that were complementary to sequences upstream (KGL-F) and downstream of Jκ1 (KGL-R) (29). Germline levels were normalized to the levels of a β-actin genomic region. The percentage of Igκ germline alleles was calculated by dividing the corresponding levels in wild type (WT) or knockout mice B cells by those in ES cells. Total RNA was extracted from 1 × 106 MoFlo sorted pre-B cells or MACS cell separation columns purified splenic B cells using TRIzol reagent (Invitrogen), to examine Igκ gene germline transcription. Then RNA was treated with DNase I and was reverse transcribed into cDNA with SuperScript III Reverse Transcriptase (Invitrogen). For real-time PCR analysis of individual Vκ gene’s germline transcripts, forward primers specific to different Vκ gene exons and a reverse primer complementary to the downstream RSS region were used (Supplemental Table I). For analysis of transcripts arising from the 5′ germline promoter upstream of the Jκ1 region, a forward 5′GT-f primer annealing immediately downstream of the promoter region and a reverse Cκ-r primer annealing in Cκ exon were used in real-time PCR assays (Supplemental Table I). Transcript levels were calculated using the ΔΔ cycle threshold method according to the manufacturer’s instructions and normalized to the cDNA levels of the mouse β-actin gene.
Three-dimensional DNA fluorescent in situ hybridization
Using our published techniques (15, 16, 30), we processed pre-B cells for three-dimensional DNA fluorescent in situ hybridization (FISH). In brief, probes for three-dimensional FISH were prepared from bacterial artificial chromosomes (BACs). We used RP23-101G13, RP23-26A6, and RP24-387E13, which correspond to the 5′, middle, and 3′ region of the Igκ locus, respectively. To make probes for each slide, we labeled 1 μg BAC DNA samples by nick translation with ChromaTide Alexa Fluor 488–5-deoxyuridine triphosphate (dUTP), ChromaTide Alexa Fluor 594–5-dUTP (Invitrogen), or Cy5-dUTP (GE Healthcare). Hybridization conditions were as described previously (15, 16, 30). FISH signals were analyzed by Leica TCS SP5 confocal microscopy with Z slice-sections separated by 0.3 μm, and the center-to-center distances between different hybridizing signals were measured using a plug-in of ImageJ software.
HS1-6−/− mice exhibit increased numbers of splenic B cells
To investigate the function of HS1-6, we generated germline-transmissible mice with a 6.3-kb deletion of HS1-6 in the endogenous Igκ locus, leaving only a single loxP site in its place. Various stages of the targeting and HS1-6 deletion were confirmed by Southern blotting (Fig. 1C, 1D, and Supplemental Fig. 1). We first investigated whether deletion of HS1-6 affected the percentages of Igκ+ B cells in spleen or bone marrow by flow cytometry. HS1-6−/− mice exhibited increased proportions of Igκ+ B cells in spleen compared with WT littermates or age-matched WT mice (53.2 ± 2.4 versus 48.1 ± 3.4%, as percentages of Igκ+ B cells among total lymphocytes, n = 10, p < 0.01, Student t test; Fig. 2A), but the percentages of Igλ+ B cells were at similar levels between HS1-6−/− and WT mice in spleen (3.1 ± 0.8 versus 3.3 ± 0.8%, n = 10, p = 0.67, Student t test; Fig. 2A). Overall, we observed an increase in total spleen cell numbers in HS1-6−/− mice compared with those of their WT littermates or age-matched WT mice (spleen cell numbers: 114 ± 27 versus 92.3 ± 19.1 × 106; n = 10, p = 0.05, Student t test), which could be almost entirely accounted for by increases in splenic B cell numbers in HS1-6−/− mice compared with those of WT (splenic B cell numbers: 65.8 ± 2.9 versus 49.2 ± 3.4 × 106; n = 6; p < 0.001, Student t test; Fig. 2B). By contrast, the percentages of Igκ+ cells in bone marrow were only slightly increased but not significantly different between HS1-6−/− and WT mice (22.0 ± 3.7 versus 19.6 ± 5.5%; n = 9; p = 0.29, Student t test; Fig. 2C). We also observed similar levels of Igλ+ cells in bone marrow in HS1-6−/− mice as compared with those of WT (1.8 ± 0.4 versus 1.8 ± 0.43%; n = 9; p = 0.83, Student t test; Fig. 2C). In addition, HS1-6−/− and WT mice exhibited no significant differences in bone marrow total cell numbers ([2 femurs]: 28.2 ± 5.2 versus 26.12 ± 4.3 × 106; n = 10; p = 0.34, Student t test). To further investigate the effect of the HS1-6 deletion on B cell development, we analyzed the subpopulations of follicular and marginal zone B cells in spleen by FACS, and found that they were all normal relative to those of WT mice (CD23+CD21lo follicular B cell: 37.1 ± 5 versus 35.7 ± 5.4%; n = 6; p = 0.65, Student t test; CD21+CD23lo marginal zone B cells: 5.7 ± 1.8 versus 5.6 ± 0.7%; n = 6; p = 0.92, Student t test; Fig. 2D). We conclude that deletion of HS1-6 causes increased numbers of splenic B cells expressing Igκ on their cell surface, but no significant defects in B cell development.
HS1-6−/− mice maintain L chain isotype exclusion, allelic exclusion, and overall levels of Vκ gene recombination
The percentages of Igκ+Igλ+ double-positive cells were the same between HS1-6−/− and WT mice, indicating that Ig L chain isotype exclusion was still intact (Fig. 3A). (Approximately 10% of the B220+ cells were neither κ+ nor λ+ and likely include NK and other cells not recognized by the Abs used in our study.) To further characterize the effects of this deletion, we bred HS1-6+/− mice with a line carrying a human Cκ knock-in allele to obtain Igκm/h and IgκΔHS1-6m/h heterozygotes (2). We found that anti-mouse Igκ (mCκ) and anti-human Igκ (hCκ) alleles were equally used in Igκm/h heterozygotes as reported previously (Fig. 3B) (2). (The 8–9% double-negative cells in Fig. 3B likely correspond to Igλ+ cells.) However, in mice bearing a deletion in HS1-6, mCκ alleles exhibited a significant preference to be used in the heterozygote splenic cells (Fig. 3B). These results are consistent with the observed increase in Igκ+ B cells in spleen from HS1-6−/− mice (Fig. 2A, 2B), and indicate that the corresponding deleted alleles gain an edge in recombination frequency compared with hCκ alleles. We also found that the heterozygotes from these groups exhibited similar levels of mCκ+hCκ+ double-positive cells (Fig. 3B), suggesting that HS1-6 deletion did not affect allelic exclusion. To further investigate the allelic exclusion issue, we performed real-time PCR analyses of germline DNA content in both pre-B and splenic B cells from WT and HS1-6−/− mice. From these results, which revealed no significant differences in total Igκ allele rearrangement (Fig. 3C), together with those from the mouse/human heterozygote studies (Fig. 3B), we conclude that allelic exclusion is fully obeyed in HS1-6−/− mice.
Deletion of HS1-6 dramatically increases Jκ-proximal Vκ gene usage and essentially eliminates usage of middle and distal Vκ genes
Previously, we demonstrated that deletion of HS1-2 (Cer) and HS3-6 (Sis) caused increased Jκ-proximal Vκ gene usage during Vκ–Jκ rearrangement in pre-B cells (15, 16). To determine whether deletion of HS1-6 also altered primary Vκ gene usage, we cloned and sequenced Vκ–Jκ1 rearrangement products from WT and mutant mice pre-B cells. As shown in Fig. 4A, Vκ genes within the first 0.2-Mb interval closest to the Jκ region were almost exclusively used in pre-B cells from HS1-6−/− mice, accounting for 95.7% of the total Vκ–Jκ1 gene usage, whereas in WT pre-B cells, Vκ genes within this corresponding 0.2-Mb interval accounted for only 10.5% of the total Vκ–Jκ1 gene usage. This pattern of preferential usage of proximal Vκ genes in HS1-6−/− mice relative to that observed in WT animals was highly statically significant: (0.2 Mb proximal Vκ gene usage versus 3.0 Mb middle and distal Vκ gene usage: 95.7 versus 4.3 in HS1-6−/− mice; 10.5 versus 89.5 in WT mice, p = e<−28, χ2 test). Interestingly, all 12 Vκ genes within the first 0.2-Mb interval belong to the Vκ21 gene family, and only 2 of these 12 genes, Vκ21-7 and Vκ21-2, contributed to 60% of the total Vκ gene usage in HS1-6−/− pre-B cells (Table I). We also cloned and sequenced Vκ–Jκ5 rearrangement products from WT and mutant mice pre-B cells. As shown in Fig. 4B, Vκ genes within the first 0.2-Mb interval were also heavily used in pre-B cells from HS1-6−/− mice, accounting for 59.3% of the total Vκ–Jκ5 gene usage instead of only 6.4% in WT pre-B cells, differences that also proved to be highly statistically significant: (0.2 Mb proximal Vκ gene usage versus 3.0 Mb middle and distal Vκ gene usage: 59.3 versus 40.7 in HS1-6−/− mice, 6.4 versus 93.6 in WT mice; p = 4.9e−27, χ2 test). To verify these results, we used real-time PCR to quantitate relative Vκ–Jκ1 gene rearrangement levels in both pre-B and splenic B cell samples from WT and HS1-6−/− mice for several individual Vκ genes and obtained very similar results to those described earlier (Fig. 4C, 4D). To determine whether the Jκ region usage was altered in HS1-6−/− mice, we used a semiquantitative PCR assay with genomic DNA isolated from pre-B cells that gives rise to four distinct bands representing V-Jκ1, V-Jκ2, V-Jκ4, and V-Jκ5 recombination products (Fig. 4E). Examination of the relative Jκ usage in pre-B cells revealed that HS1-6−/− mice still used all four Jκ regions in Vκ–Jκ joining, although the Jκ1 usage in HS1-6−/− mice appeared to be slightly increased compared with that of WT mice as revealed by PhosphorImager analysis of the Southern blots (Jκ1 usage: 1.56 versus 1; Jκ2 usage: 0.77 versus 1; Jκ4 usage: 0.96 versus 1; Jκ5 usage: 0.98 versus 1). Finally, to investigate the developmental timing and the fidelity of rearrangement in pre-B cells, we accessed the extent of insertion of N nucleotides in Vκ–Jκ recombination junctions in pre-B cells, as well as the extent of which various recombination junctions were in frame for uninterrupted translation. In contrast with P nucleotides, which become inserted naturally by the recombination mechanism, N nucleotide insertions require TdT activity, which is normally expressed in pro-B, but not in pre-B, cells when Igκ genes normally rearrange (31). We found no major differences in the percentages of N and P nucleotides for the Vκ–Jκ recombination junctions in pre-B samples from WT and HS1-6−/− mice (Fig. 4F). When we considered the in-frameness ratio for all Vκ–Jκ1 recombination junctions, our results revealed higher levels in HS1-6−/− pre-B cells compared with those of WT (in-frameness versus out-of-frameness: 46 versus 54 in HS1-6−/− mice; 31 versus 69 in WT mice; p < 0.001, χ2 test); however, this observation is likely because Vκ21 gene family members are most heavily used in HS1-6−/− pre-B cells, and these genes exhibit higher in-frameness recombination ratios relative to all Vκ genes even in WT animals (Fig. 4F). Collectively, we conclude that HSs within the Vκ–Jκ intervening region dramatically regulate Vκ gene usage without significantly changing Jκ usage or the developmental timing and fidelity of rearrangement.
|.||% Vκ Gene Usage in WT Pre-B Cells .||% Vκ Gene Usage in HS1-6−/− Pre-B Cells .|
|.||% Vκ Gene Usage in WT Pre-B Cells .||% Vκ Gene Usage in HS1-6−/− Pre-B Cells .|
Detailed representation of the Vκ–Jκ1 rearrangement products of pre-B cells from WT and HS1-6−/− mice shown in Fig. 4A. Vκ gene percent usages relative to the total Vκ gene usage as 100% are presented.
Pre-B and splenic B cells from HS1-6−/− mice exhibit massive levels of germline proximal Vκ gene transcripts
Germline transcription of the Igκ locus has long been thought to target the recombinase apparatus (32–35). To address whether the dramatically increased proximal Vκ gene usage correlated with increased proximal Vκ gene germline transcription, we measured by real-time PCR the levels of germline transcripts in pre-B cells arising from selected Vκ genes representing diverse physical positions in the locus and from the 5′-promoter upstream of the Jκ region (5′GL) in samples from WT and mutant mice. Remarkably, the levels of proximal Vκ21 gene germline transcripts in HS1-6−/− mice strikingly increased >20-fold over those of WT in pre-B cells, whereas 5′GL and those of distal Vκ genes exhibited only minor differences (Fig. 5A). Interestingly, these hyperelevated germline transcription levels of proximal Vκ genes were also observed in splenic B cells (Fig. 5B). Indeed, as an unpredictable consequence of this deletion, we observed pronounced and strikingly increased levels of unrearranged proximal Vκ gene transcripts, in both pre-B and splenic B cells.
Deletion of HS1-6 results in proximal Vκ gene rearrangement in T cells
Previously we found that Igκ gene rearrangements were detectable in CD4+CD8+ DPT cells from HS1-2−/− (Cer−/−) mice (16). As expected, HS1-6−/− mice also exhibited Igκ gene rearrangement in T cells, but not in their WT counterparts, and these rearrangements modestly favored Jκ1 and Jκ2 usage (Fig. 6A). Characterization of these recombination products by cloning and sequencing revealed that proximal Vκ genes were preferentially used just as seen in pre-B cells (Fig. 6B). To validate that rearrangement indeed occurred in T cells of the mutant mice, we accessed the extent of insertion of N nucleotides in Vκ–Jκ recombination junctions in these cells. As described earlier, N nucleotide insertions require TdT activity, which is also normally expressed in T cells, but not in pre-B cells (31). In marked contrast with the 4% level of N regions seen in pre-B cell samples from these mutant mice (Fig. 4F), we found that 69% of Vκ–Jκ junctions had N nucleotide insertions in CD4+CD8+ T cell samples from HS1-6−/− mice (Fig. 6C), providing persuasive evidence that these T cells indeed rearranged Igκ genes. However, a FACS analysis of T cell development revealed no obvious deleterious effects of such Igκ gene rearrangements in these cells (Fig. 6D).
Igκ gene locus contraction is reduced in pre-B cells from HS1-6−/− mice
We have previously demonstrated that proximal Vκ gene usage is also dramatically favored in HS1-2−/− mice in which Igκ locus contraction is reduced in pre-B cells (16). To verify our expectation that locus contraction is also reduced in pre-B cells from HS1-6−/− mice, we performed three-dimensional DNA FISH experiments using Igκ gene BAC probes corresponding to 5′, middle, and 3′ locations in the locus (Fig. 7A, probes A, B, and C, respectively). As shown in Fig. 7B, representative confocal images generated using these probes revealed as expected that pre-B cell nuclei from WT mice exhibited looped and contracted Igκ gene structures in agreement with a previous report (36), whereas corresponding samples from HS1-6−/− mice exhibited less-contracted, nonlooped patterns similar to HS1-2−/− (Cer−/−) mice. To quantitate these results, we measured the center-to-center distances between A and B, B and C, and A and C hybridization signals for several hundred alleles in these samples. As shown in Fig. 7C, contraction was statistically significantly decreased between chromosome regions corresponding to hybridization signals B and C and A and C by the HS1-6 deletion, but not significantly between the regions corresponding to hybridization signals A and B. The mean distances between respective A and B, B and C, and A and C hybridization signals were 0.316-, 0.326-, and 0.279-μm for WT samples and 0.35-, 0.45-, and 0.472-μm for HS1-6−/− samples (Fig. 7C, dashed lines in the boxes). These differences correspond to 10, 38, and 69% decreases in contraction in the respective 5′- and 3′-halves of the locus, or for the locus as a whole, for HS1-6−/− mice samples in comparison with those from WT mice.
We have previously shown that HS1-6 within the 20-kb Vκ–Jκ intervening sequence can be functionally subdivided into HS1-2 and HS3-6, which respectively comprise elements specifying locus contraction (Cer) and recombination silencing (Sis) in pre-B cells (15, 16). In this study, we have deleted both Cer and Sis to investigate whether their combined deletion results in the predictable sum of their individual single-deletion phenotypes and whether any new phenotypes may result because of their potential functional redundancies. Our results both strengthen and extend the conclusions of these previous studies.
Overall, locus contraction in pre-B cells was reduced in HS1-6−/− mice, but within statistically similar levels compared with those exhibited previously by Cer−/− mice (16). Moreover, although HS1-6−/− mice exhibit more dramatic preferential use of proximal Vκ genes in comparison with our earlier results with either Cer−/− or Sis−/− mice (15, 16), such usage is only modestly more extreme than that exhibited by Cer−/− mice per se (16). Likewise, HS1-6−/− mice exhibit Igκ rearrangement in T cells, a phenotype previously observed in Cer−/− mice (16). Although such nonlineage-specific Igκ rearrangement did not affect the final outcome of CD4 and CD8 T cell populations as revealed by FACS, in the future, more detailed studies on the earlier stages of T cell development encompassing the DN pre-TCR checkpoint in these mutant cells may reveal more significant T cell population perturbations.
Importantly, HS1-6−/− mice exhibited a novel phenotype not seen in our previous studies, namely, a pronounced and striking increase in proximal Vκ gene germline transcription, in both pre-B and splenic B cells. These results suggest that Cer and Sis may be functionally redundant with respect to buffering proximal Vκ gene germline transcription, because our previous studies have shown that such germline transcription is only modestly upregulated upon single deletion of either element (15, 16). However, our results reveal that upon deletion of both elements together with ∼2 kb of additional intervening sequence, Vκ21-7 gene germline transcription increased >20-fold in pre-B cells and up to nearly 60-fold in splenic B cells over that of WT controls. Interestingly, both Cer and Sis have pairs of CCCTC-binding factor (CTCF) binding sites that are occupied by the protein in pre-B cells (16). CTCF has been implicated in numerous functions including long-range chromosomal looping and enhancer insulator activities (37). Our results suggest that either Cer or Sis may act as insulator boundaries by preventing the downstream enhancers intronic enhancer, E3′, and Ed from activating proximal Vκ gene germline transcription, whereas their double deletion may relieve this block and lead to massive Vκ21 gene germline transcription. Significantly, ∼40% of splenic B cells will possess one productively rearranged Igκ allele and one unrearranged allele in germline status (26). Therefore, we conclude that another function of the Vκ–Jκ intervening region is to regulate the massive overproduction of noncoding transcripts in normal B cells by silencing the transcription of unrearranged proximal Vκ genes. We predict that such futile germline transcription will have important deleterious outcomes in B cell functions, which remain to be discovered in the future.
It has long been thought that germline transcription is linked to targeting the recombination apparatus during V(D)J joining (32–35). However, although the striking increase in proximal Vκ gene germline transcription in HS1-6−/− mice correlates with the dramatic preferential use of these segments in recombination, no such increase in germline transcription occurs in HS1-2 (Cer−/−) mice that nearly mimic the same preferential usage of these Vκ genes (16). Rather, the reduced locus contraction seen in pre-B cells from both of these knockout mice lines best correlates with the observed dramatic increase in proximal Vκ gene usage in recombination.
In the mouse Igh locus, a regulatory region termed IGCR1 within the Vh-Dh intervening region may share certain functional features with HS1-6 in the Igκ locus. Deletion of the IGCR1 or mutation of its CTCF binding sites cause upregulated proximal Vh gene germline transcription and a skewed IgH Ab repertoire because of markedly increased Dh proximal Vh gene rearrangements (38). Both HS1-6 and IGCR1 bind CTCF, so similar roles of these elements may be that they act as insulator or boundary elements to prevent the downstream enhancers in their corresponding loci from generating active chromatin conformations to their proximal V genes (16, 38). In addition, deletion of either IGCR1 or Cer/HS1-6 results in nonlineage-specific V(D)J rearrangements in DPT cells (16, 38). However, Cer−/− mice exhibit a strikingly skewed Vκ gene usage that is similar to HS1-6−/− mice without any significant increase in proximal Vκ gene germline transcription (16). This suggests that Cer controlled Igκ locus contraction plays a crucial role in regulating proximal versus distal Vκ gene usage. In contrast with the Cer or HS1-6 deletion, the IGCR1 deletion does not affect global Igh long-range interactions or locus contraction (39). These observations together allow the conclusions that increased proximal V gene rearrangements can be triggered by either increased local germline transcription or reduced long-range locus contraction. Whether CTCF and/or other Cer binding proteins control Igκ locus contraction is still an open question for future investigation.
We are indebted to Michel C. Nussenzweig of The Rockefeller University for kindly providing mice. We thank Jose Cabrera for expert graphic illustrations.
This work was supported by the National Institutes of Health (Grants GM29935 and AI067906), the Robert A. Welch Foundation (Grant I-0823 to W.T.G.), and the National Natural Science Foundation of China (Grant 31270928 to Y.X.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
bacterial artificial chromosome
fluorescent in situ hybridization
DNase I hypersensitive site
The authors have no financial conflicts of interest.