Identification of the USP21SV without Nuclear Export Signal.

<p>A) Total RNA from 10-week-old mouse liver was analyzed by RT-PCR using USP21 gene-specific primers. Two different sized PCR products were detected as denoted by the arrow. B) Design of the specific primers located in the USP21 gene. C) Schemes of USP21LV and USP21SV. The alternatively spliced sequence in USP21SV is indicated with the blue box. D) Coomassie staining of Usp21SV and Usp21 LV.</p

ubH2A signal decreased when both USP21 variants are over-expressed.

<p>Hela cells were transfected either with EGFP-USP21SV (A–H, M–P) or EGFP-USP21LV (I–L, Q–T) for 24 hours. Anti-lamin antibody was used for immunofluorescence identification of the nucleus (A, E, I) and anti-tubulin antibody was used for immunofluorescence identification of the cytoplasm (M, Q). Anti ubH2A antibody was used to evaluate nuclear ubH2A (G, K, O, S). Control IgG was used as a negative control for immunofluorescence of ubH2A (C). The merged image illustrates the relationship of either EGFP-USP21SV (H, P) or EGFP-USP21LV (L, T) expression and ubH2A. Merging of Control and GFP showed features of the cell with GFP but without H2A ubiquitylation (D). Scale bar indicated 50 μm.</p

USP21SV is more active than USP 21LV in vivo.

<p>293T cells were transfected either with EGFP-USP21SV, EGFP-USP21LV or EGFP as indicated. Lipofectamine LTX & PLUS treatment was used as a control. After 24 hours cells were subjected Western blot analysis. A) Anti-EGFP was used for assessment of USP21 introduction and amido black was used for loading control. Quantification of ubH2A was used to evaluate activity of USP21 variants <i>in vitro</i>. B) Intensity of ubH2A was measured using three independent transfection experiments. The intensity of ubH2A is in arbitrary units compared to the value of the control.</p

Sequences of the long and short variant of USP21. Nuclear export signal.

<p>(NES) is indicated with a red asterisk. USP21SV is deficient in part of exon 2, comprising an 87 amino acid sequence with a bold italic blue letter between two arrowheads. Active site residues are indicated with a bold red letter. Estimated microtubule binding sites are indicated with a bold italic purple letter.</p

USP21LV localizes mainly in the cytoplasm.

<p>Detailed analysis of box in Fig.–F and G–L respectively. Merger of B & D, C & D, H & J, I & J is shown in E, F, K, L, respectively. Scale bar indicated 10 μm.</p

Both USP21SV and USP21LV deubiquitylate ubH2A and activate transcription.

<p>A) Deubiquitylation assay. Different amounts of recombinant USP21SV and USP21LV were incubated with native chromatin as indicated. Signals from ubH2A were detected by Western blotting using anti-ubH2A and Alexa 647-Protein A (Life Technologies). Amido black staining was used as loading control. B) Native H3-H4 and ubH2A-H2B were purified from mouse liver. C) Chromatin was assembled by salt dialysis and digested with micrococcal nuclease. D) Ubiquitylated chromatin was subjected to transcription after treatment with USP21-SV, USP21-LV or control buffer as indicated. Deubiquitylated H2A was confirmed by Western blotting with anti ubH2A. Transcripts were detected by primer extension.</p

Loss of Pcgf5 Affects Global H2A Monoubiquitination but Not the Function of Hematopoietic Stem and Progenitor Cells

<div><p>Polycomb-group RING finger proteins (Pcgf1-Pcgf6) are components of Polycomb repressive complex 1 (PRC1)-related complexes that catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1), an epigenetic mark associated with repression of genes. Pcgf5 has been characterized as a component of PRC1.5, one of the non-canonical PRC1, consisting of Ring1a/b, Rybp/Yaf2 and Auts2. However, the biological functions of Pcgf5 have not yet been identified. Here we analyzed the impact of the deletion of <i>Pcgf5</i> specifically in hematopoietic stem and progenitor cells (HSPCs). <i>Pcgf5</i> is expressed preferentially in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) compared with committed myeloid progenitors and differentiated cells. We transplanted bone marrow (BM) cells from <i>Rosa</i>::<i>Cre-ERT</i> control and <i>Cre-ERT;Pcgf5</i>^<i>fl/fl</i> mice into lethally irradiated recipient mice. At 4 weeks post-transplantation, we deleted <i>Pcgf5</i> by injecting tamoxifen, however, no obvious changes in hematopoiesis were detected including the number of HSPCs during a long-term observation period following the deletion. Competitive BM repopulating assays revealed normal repopulating capacity of <i>Pcgf5</i>-deficient HSCs. Nevertheless, <i>Pcgf5</i>-deficient HSPCs showed a significant reduction in H2AK119ub1 levels compared with the control. ChIP-sequence analysis confirmed the reduction in H2AK119ub1 levels, but revealed no significant association of changes in H2AK119ub1 levels with gene expression levels. Our findings demonstrate that Pcgf5-containing PRC1 functions as a histone modifier <i>in vivo</i>, but its role in HSPCs is limited and can be compensated by other PRC1-related complexes in HSPCs.</p></div

Undisturbed TCRα chain joining region usage in newly generated <i>Traj18</i>-deficient mice as revealed by next generation sequencing.

<p>Sequencing of TCRα chain joining region. PCR was carried out to amplify <i>Trav11-Trac</i> transcripts using cDNA prepared from sorted TCRβ^low CD4⁺CD8⁺ double-positive thymocytes from <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- (red bars) and <i>Traj18</i>^-/- (blue bars) mice. Bars depict mean ± SEM percentages of productive <i>Traj</i> gene segment rearrangements, and data are derived from three biologically independent samples per genotype. Numbers in parenthesis indicate the total number of sequences analyzed. The data are from one experiment.</p

Newly generated <i>Traj18</i>-deficient mice lack Vα14 NKT cells.

<p>(A) Flow cytometry profiles of thymocytes, splenocytes and liver mononuclear cells from WT, <i>Traj18</i>^-/- and <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- mice. Unloaded CD1d dimer staining was used as a staining control. Numbers depict percentage of αGC/CD1d dimer⁺ TCRβ⁺ NKT cells among viable CD8^-B220^- gated lymphocytes. The data are representative of three independent experiments. (B) <i>In vivo</i> cytokine production by NKT cells upon systemic activation with αGalCer administration. WT or <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- or <i>Traj18</i>^-/- mice were injected intravenously with 2 μg of αGalCer and blood plasma were collected after either 3 h and 24 h, and IFN-γ and IL-4 concentrations were measured using cytokine beads assay. Bars depict mean ± SEM of <i>n</i> = 3 mice per genotype analyzed. Data are representative of three experiments.</p

Generation of novel <i>Traj18</i>-deficient mice.

<p>Schematic representation of a <i>Traj18</i> region targeting construct, <i>Traj18</i> region before and after homologous recombination, and the genomic locus after FLP- and Cre-mediated deletions of the neomycin resistance gene and <i>Traj18</i>, respectively.</p