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

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

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

Over-expression of miR290-5p or miR292-5p induces activation of E2A and NF-κB.

<p>4A. ChIP analysis of HF4 AMuLV cells expressing His-FLAG-E2A, at endogenous levels, and over-expressing either miR129-2_3p, miR290-5p, or mir292-5p. Chromatin samples were immunoprecipitated with anti-FLAG or IgG control antibody. Relative enrichment of bound DNA over input was determined by subjecting precipitates to qPCR with primers specific to the Eκi binding region for E2A. This data shows one representative experiment of two independent experiments. 4B. ChIP analysis of E2A+/+ AMuLV cells over-expressing either miR129-2_3p, miR290-5p, or mir292-5p, in the absence or presence of STI571 (1 µM, 16 hr). Chromatin samples were immunoprecipitated with anti-p50 or IgG control antibody. Relative enrichment of bound DNA over input was determined by subjecting precipitates to qPCR with primers specific to the Eκi binding region for NF-κB. This data shows one representative experiment of two independent experiments. 4C. Immunoblot analysis of E2A+/+ AMuLV cells expressing either an empty vector control, miR290-5p, or miR292-5p and cultured in either the absence or presence of STI571 (2.5 µM, 12 hr). Immunoblot was probed with anti-IκBα and anti-Tubulin antibodies. 4D. Luciferase assay of total cell lysates from HEK293 cells transiently transfected with either a wild-type IκBα 3′UTR reporter or a mutant IκBα 3′UTR reporter along with a scrambled miRNA, mir290-5p or miR292-5p. Error bars represent range for biological replicate luciferase reactions. Data shows one representative experiment of at least three 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

Skewed hematopoietic vs. endothelial potential from EB-derived CD34⁺CD45⁻ cells.

<p>(A) The indicated hESC lines were first differentiated as EBs in EB media without lineage skewing cytokines. CD34⁺CD45⁻ cells were enriched by fluorescence activated cell sorting (FACS) on day 10 of EB culture and differentiated on fibronectin-coated plates in the presence of IL-3, IL-6, SCF, G-CSF, Flt3L, and BMP4 for an additional 7 days. Representative flow cytometry plots of CD45 (hematopoietic marker) and VE-cadherin (endothelial marker) are presented. (B) Comparative analysis of endothelial potential from independently-derived hESC lines. Endothelial differentiation of hESC lines was determined by a two-step culture. hESCs were initially differentiated for 9 days as EBs as in (A), and CD34⁺ cells enriched by FACS. CD34⁺ cells were plated on fibronectin-coated plates in the presence of an endothelial growth factor cocktail containing bovine pituitary extract, heparin, and hVEGF, and analyzed after 7 additional days in culture. Representative flow cytometry plots of CD45 and VE-cadherin are presented. (C) Graphs depict the relative number of hematopoietic (CD45⁺) or (D) endothelial lineage (VE-cadherin⁺) cells as identified by flow cytometry over the starting (CD34⁺, CD45⁻, VE-cadherin⁻) population. Three independent experiments are shown in (C) and (D). The right panels denote the average of the three independent data sets with error bars and standard deviation between hESC lines. * denotes p<0.05.</p

Comparative analysis of hemangioblast development from independently-derived hESC lines.

<p>Several hESC lines were differentiated as embryoid bodies (EB) for nine days after several trypsin passages (A, top panel) or after several manual passages (A, middle panel) in EB media without lineage-skewing cytokines. CD34⁺ and CD34⁺CD45⁺ development was determined by flow cytometric analysis of several cell surface markers indicative of differentiation state. The proportion of hESC-derived CD34⁺CD45⁻ cells is presented on differentiating hESCs in black. The proportion of CD34⁺CD45⁺ progenitors is indicated in white. HuES8, HuES14, and HuES15 cell lines were highly susceptible to gross karyotypic abnormalities during trypsin passage (as indicated). H1, H9, and HSF6 manually passaged cells had previously been passaged with trypsin (>5 manual passages before differentiation). (A, bottom panel) Independently-derived hESC were differentiated on an OP9 monolayer for nine days, and CD34 and CD45 cell surface expression analyzed by flow cytometry. Two representative experiments of each condition are presented. (B) Abnormal karyotypes observed in trypsin passaged cells. (C) Representative time course of CD34 expression on manually passaged, independently-derived hESCs differentiated as EBs or on an OP9 monolayer. CD34 expression was analyzed on days 3, 6, 9, 12, 15, 18.</p