Deletion of <i>Gfi1</i> and <i>Gfi1b</i> results in increased V(D)J recombination in B cells and pDCs.

<p>(<b>A</b>) Flow cytometric analysis of VEX expression in B cells derived from the bone marrow of 2 individual <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre, SVEX</i> mice cultured in 5ng/ml IL-7 for 7 days (solid line), untreated (top panel) and treated (bottom panel) with tamoxifen (4-OHT). Cells were gated on B220⁺. Shaded histogram denotes background fluorescence from <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> cells. Vertical axis ('% of max') indicates a scale of relative cell numbers with the median value set as 100%. (<b>B</b>) Flow cytometric analysis of VEX expression in pDCs derived from bone marrow from 2 individual <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre, SVEX</i> mice cultured in 25ng/ml Flt-3L for 8 days (solid line), untreated (top panel) and treated (bottom panel) with tamoxifen. Cells were gated on B220⁺ CD11c⁺ cells. Shaded histogram denotes background fluorescence from <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> cells. Vertical axis ('% of max') indicates a scale of relative cell numbers with the median value set as 100%. All data are representative of at least three independent experiments. (<b>C</b>) Dot plot showing percentage of SVEX+ cells in Flt-3L cultures untreated (-4-OHT) and treated (+4-OHT) with tamoxifen. p-value was calculated using the two-tail paired Student’s <i>t</i>-test.</p

Deleting <i>Gfi1</i> and <i>Gfi1b</i> does not result in a change in lymphoid- or pDC-specific genes in pDCs.

<p>(<b>A</b>) RT-PCR of lymphoid-specific gene expression in <i>ex </i><i>vivo</i> differentiated B220⁺ CD11c⁺ pDCs from <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> mice untreated (WT) and treated (KO) with tamoxifen. RNA isolated from primary B (B) or T (T) cells were used as controls. (<b>B</b>) Quantitative RT-PCR of pDC-specific genes in <i>ex </i><i>vivo</i> differentiated B220⁺ CD11c⁺ pDCs from <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> mice untreated (WT) and treated (KO) with tamoxifen. Expression values in arbitrary units are normalized to <i>Hprt</i> transcript abundance.</p

Deletion of <i>Gfi1</i> and <i>Gfi1b</i> results in increased expression of <i>Rag</i> in pDCs.

<p>(<b>A</b>) Quantitative RT-PCR analysis of <i>Rag1</i> transcript levels in sorted B220⁺ CD11c⁺ pDCs derived from 3 individual <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> mice untreated and treated with tamoxifen (4-OHT). Values are normalized to <i>Hprt1</i> transcript abundance. p-values were calculated with the two-tail Student’s <i>t</i>-test. * denotes p < 0.005. (<b>B</b>) Genotyping PCR of <i>Gfi1</i> and <i>Gfi1b</i> loci from sorted B220⁺ CD11c⁺ pDCs derived from 4 individual <i>Gfi1</i>^<i>f/f</i><i>; Gfi1b</i>^<i>f/f</i><i>; ERCre</i> mice untreated and treated with tamoxifen (4-OHT). Genomic DNA was isolated and subjected to PCR analysis using primers that detect wildtype (WT), floxed and deleted alleles of <i>Gfi1</i> and <i>Gfi1b</i>. PCR products were run on 1% agarose gel and visualized with ethidium bromide. All data are representative of at least two independent experiments.</p

The Proximal J Kappa Germline-Transcript Promoter Facilitates Receptor Editing through Control of Ordered Recombination

<div><p>V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first <i>cis</i>-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.</p></div

miR290-5p/292-5p Activate the Immunoglobulin <em>kappa</em> Locus in B Cell Development

<div><p>Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the <em>kappa</em> intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to <em>kappa</em> locus regulatory sequences.</p> </div

The proximal Jκ GT promoter controls Jκ choice.

<p><b>A)</b> LM-PCR detects total DNA breaks at Jκ gene segments in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>B)</b> LM-PCR detects premature DNA breaks at Jκ2, Jκ4, and Jκ5 in pre-B cells from wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Linker ligated genomic DNA was first amplified with several Jκ-specific forward primers (FP) and a linker-specific reverse primer (LP) and then hybridized with Jκ RSS probes. Results are representative of at least two independent experiments. <b>C)</b> VJ coding joint PCR detects individual Jκ segments in completed VκJκ joints in B cells from bone marrow or spleen of wildtype mice (wt) or mice lacking the proximal GT promoter (κD, deletion; κS, stuffer). Genomic DNA was first amplified with a degenerate Vκ-specific forward primer and a reverse primer (MAR35) that binds downstream of Jκ5 and then hybridized with a probe (5’MAR35) that binds downstream of Jκ5 but upstream of the reverse primer. Results are representative of at least two independent experiments. </p

κGT induction is blunted upon knockdown of miR290-5p or miR292-5p.

<p>3A. qPCR analysis of κGT expression in RNA purified from E2A+/+ AMuLV cells expressing a miR290-5p or miR292-5p sponge knockdown construct, and cultured in the presence of STI571 (2.5 µM) for the indicated lengths of time. Data was normalized to the expression of <i>Hprt</i>. Error bars represent range for replicate qPCR reactions. Asterisk represents P value <0.05. The P value was derived by the Student’s T test. These data are from one representative experiment of at least four independently performed experiments. 3B, C. qPCR analysis of (B) κGT or (C) Rag1 expression in RNA purified from wild-type primary pro-B cells transduced with either a miR129-2_3p control knockdown sponge, or a miR290-5p or miR292-5p knockdown sponge. Wild-type primary pro-B cells were cultured in rIL7 (2 ng/ml) for 5 days and then transduced with indicated sponge construct. Marker positive cells were then sorted and IL7 withdrawn from culture for 24 hrs before RNA was harvested. qPCR measures (B) κGT or (C) Rag1 expression in cells before or after the withdrawal of IL7. Data was normalized to <i>Hprt</i> expression levels. Error bars represent range for replicate qPCR reactions. Asterisk represents P value <0.05. The P value was derived by the Student’s T test. These data show one representative experiment of at least four independently performed experiments.</p

miR290-5p and miR292-5p are induced at the pro-B to pre-B transition.

<p>1A. Heat-map representing levels of miRNAs from a microarray analysis of RNA purified from the indicated AMuLV cell lines cultured in the absence or presence of STI571 (2.5 µM, 12 hr). Performed once in three independently transformed cell lines. 1B. Schematic depiction of the shared seed sequence of the mature miR290-5p and miR292-5p microRNAs. 1C. qPCR analysis of miR290-5p or miR292-5p expression levels in RNA purified from E2A+/+ AMuLV cells cultured in the absence or presence of STI571 (2.5 µM, 12 hr). Data was normalized to the expression of miR129-2_3p. Error bars represent range for replicate qPCR reactions. The data shown is from one representative experiment of three biological replicates. 1D. qPCR analysis of miR290-5p or miR292-5p in primary wild-type pro-B (B220+, CD43+, IgM−) cells or pre-B (B220+, CD43−, IgM−) cells. Data was normalized to the expression of miR129-2_3p. Error bars represent range for replicate qPCR reactions. Data shown is from one representative experiment, of three independent sort experiments.</p

The proximal Jκ GT promoter is inactive prior to Igκ recombination.

<p><b>A)</b> Flow cytometry detects expression of κGFP (proximal GT promoter reporter; top) and κhCD4 (distal GT promoter reporter; bottom) in RAG-deficient developing B cells carrying either no transgene, a B1-8^wt HC transgene, or a B1-8^wt HC transgene plus a κHEL transgene. Pro-B and pre-B cells are gated B220⁺ IgM⁻, immature (imm) B cells are gated B220⁺ IgM⁺ IgD⁻, transitional (trans) B cells are gated B220⁺ IgM⁺ IgD^low, and mature (mat) B cells are gated B220⁺ IgM⁺ IgD^high. Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>B)</b> Flow cytometry detects expression of κGFP (top) and κhCD4 (bottom) in non-editing (B1-8^wtHC/κHEL/RAG−/−) or receptor-editing (B1-8^lowHC/κHEL/RAG−/−) B cells (gated B220+ IgM−). Grey shaded histograms show cells from a C57Bl/6 control mouse. Results are representative of at least two independent experiments. <b>C)</b> Northern blotting of Jκ GTs in an Abelson virus-transformed pre-B cell line treated with either STI-571 (STI, which mimics pre-BCR signaling) or the TLR4 ligand LPS. mRNA was hybridized with a Cκ-specific probe (top) that recognizes mature Igκ transcripts, distal GTs, and proximal GTs, the latter of which can be identified by their smaller size. Additionally, the blot was hybridized with a probe specific for distal GTs (middle). Beta-tubulin transcripts (bottom) served as a loading control. Results are representative of two independent experiments. </p

Germline knockout of the miR290 cluster affects the pre-B cell population.

<p>5A. FACS analysis of 6 week old miR290 cluster knockout or wild-type mice. FACS plot reflects CD19 enriched cells gated on IgM negative cells. B220+, CD43+ correspond to pro-B cells and B220+, CD43− correspond to pre-B cells.; WT: pro-B, 5.72%, pre-B 84.1%; KO: pro-B, 2.47%, pre-B 91.1%. This experiment represents one of four independent experiments. Each experiment was with at least one mouse per genotype. 5B. Plot of the percentages of CD19+, IgM− pro-B (B220+, CD43+) and pre-B (B220+, CD43−) for each independent knockout and wild-type mouse analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043805#pone-0043805-g005" target="_blank">Figure 5A</a>. Average percentages are WT: pro-B, 6.05%, pre-B 85.3%; KO: pro-B, 3.4%, pre-B 90.6%. Line represents the average percentage for each population and the P value was derived by the Student’s T test. The average was derived from at least four mice per genotype. 5C, D qPCR analysis of (C) κGT or (D) Rag1 levels in RNA purified from flow-sorted pro-B (B220+, CD43+, IgM−), large pre-B (B220+, CD43−, IgM−, FSC-Hi), and small pre-B (B220+, CD43−, IgM−, FSC-Lo) cells. Mice were 6 week old miR290 cluster knockout or wild-type mice. The data was normalized to <i>Hprt</i> expression. Error bars represent range for replicate qPCR reactions. Here we show qPCR data from a representative mouse from each genotype. This experiment has been repeated with four mice per genotype and data shown is representative of three out of four miR290 knockout mice.</p