Regulation of B-cell development and tolerance by different members of the miR-17∼1/492 family microRNAs

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼1/492 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathwaysThis study is supported by the PEW Charitable Trusts, Cancer Research Institute, Lupus Research Institute and National Institute of Health (R01AI087634, R01AI089854, R56AI110403 and R56AI121155 to C.X.

Differential sensitivity of target genes to translational repression by miR-17~92

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genesCX is a Pew Scholar in Biomedical Sciences. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, National Institute of Health (R01AI087634, R01AI089854, RC1CA146299, R56AI110403, and R01AI121155 to CX), National Natural Science Foundation of China (31570882 to WHL, 31570883 to NX, 31570911 to GF, 91429301 to JH, 31671428 and 31500665 to YZ), 1000 Young Talents Program of China (K08008 to NX), 100 Talents Program of The Chinese Academy of Sciences (YZ), National Program on Key Basic Research Project of China (2016YFA0501900 to YZ), the Fundamental Research Funds for the Central Universities of China (20720150065 to NX and GF), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A1A01052387 to SGK, NRF-2016R1A4A1010115 to SGK and PHK), and 2016 Research Grant from Kangwon National University (SGK)

Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

<div><p>RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoH^tg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoH^tg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4⁺CD8⁺ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2^-/-RhoH^tg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.</p></div

Analysis of T cell development in RhoH overexpressing mice.

<p>Analysis by flow cytometry of thymocytes and splenocytes from RhoH^+/+RhoH^Tg (red) and RhoH^+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4^-CD8^-CD44^-CD25⁺ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH^+/+RhoH^Tg and RhoH^+/+ mice in either MHC^+/+ (C, n = 6) or MHC^-/- (D, n = 5) background. Solid line and dashed line represent RhoH^+/+ and RhoH^+/+RhoH^Tg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4⁺ T (F) or CD8⁺ T (G) cells gated on TCRβ⁺ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.</p

The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH^-/- mice.

<p>(A) Analysis of RhoH protein expression by western blot in RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes. (B, C) Analysis of RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4^-CD8^- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4⁺CD8⁺ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001</p

Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.

<p>(A) FACS analysis of the <i>in vitro</i> differentiation of thymocytes to the DP stage. DN3 cells from Rag2^-/-, Rag2^-/-RhoH^tg, and Rag2^+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.</p

The CABIT1 domain of Themis is important for nuclear localization.

<p>(A) DP-thymocytes from Themis^+/+ mice were fractionated into cytoplasm, membrane and nuclear fractions and immunoblotted with indicated Abs. The CRK and PARP proteins were used as purity controls for the cytoplasmic and nuclear fractions, respectively. Data are representative of more than four independent experiments. (B) Representative micrograph showing localization of Themis (white) in cytoplasm and nucleus stained with DAPI (blue), together with BF (bright field). Data are representative of three independent experiments. (C) Localization of Themis mutants was analyzed by Western blot in cytoplasmic and nuclear extracts. Thymocytes from Themis mutant mice were fractionated into the cytoplasm and nucleus. (D) Bar graph shows that the nuclear/cytoplasmic Themis protein ratio compared with Themis^+/+ mice. Data are representative of more than three independent experiments. Significant differences are noted in the graphs. *p<0.05.</p

The CABIT1 and CABIT2 in Themis have different functions.

<p>(A) CD4 and CD8 profiles of thymocytes and splenocytes of Themis^+/+, Themis^+/+ ΔCore1, and Themis^+/+ ΔCore2 mice. Numbers in plots show the frequency of cells in the indicated area. Bar graphs show absolute number of total thymocytes and thymocyte subsets from Themis^+/+, Themis^+/+ ΔCore1, and Themis^+/+ ΔCore2 mice (mean ± SEM). Data are representative of four independent experiments. (B) Proportion of post-selected CD69⁺ TCR^hi cells in CD4⁺CD8⁺ (DP) thymocytes. Data are representative of four independent experiments. (C) Surface expression of CD25 and CD44 on gated DP thymocytes. Data are representative of four independent experiments. (D) CD44 and CD62L profile of splenic CD4SP and CD8SP cells. Data are representative of four independent experiments. (E) Representative histogram overlays of phosphorylation of ERK. DP thymocytes were stimulated with anti-CD3 plus anti-CD4 Abs for 1min, then intracellular staining of phosphorylated-ERK antibody was performed (solid line). The shaded line is without stimulation. Data are representative of three independent experiments. (F) Absolute number of thymic CD4⁺25⁺Foxp3⁺ Treg cells (mean ± SEM). Data are representative of four independent experiments. Significant differences are noted in the graphs. *p<0.05, **p<0.01. N.S. = not significant.</p

Themis mutants lack tyrosine-phosphorylation and Grb2-association.

<p>(A) Analysis of tyrosine-phosphorylation and protein interactions of Themis mutants. Thymocytes from the indicated mutant mice were stimulated with anti-CD3 plus anti-CD4 antibodies. Proteins were immunoprecipitated (IP) with anti-Themis antibody and analyzed by immunoblot (IB) with the indicated antibodies. Data are representative of four independent experiments. (B) Anti-Themis monoclonal antibody (mAb) 2E7 binds to the PRS motif. Immunoprecipitated Themis proteins from Themis^+/+, Themis^−/− ΔCore1, Themis^−/− ΔCore2 and Themis^−/− ΔPRS thymocytes were immunoblotted with anti-Themis mAb 2E7. Data are representative of three independent experiments. (C) Cell lysates from Themis^+/+ thymocytes were sequentially immunoprecipitated with 2E7 and anti-Themis polyclonal antibody (pAb). Grb2 was co-precipitated with anti-Themis pAb but not with 2E7. Data are representative of three independent experiments.</p