<i>Alox5</i> involvement in hematopoietic cell self-renewal.

<p>(A) <i>Alox5</i> required for long-term self-renewal of hematopoietic cells by RE9a. Colony numbers from wildtype or <i>Alox5</i>-/- bone marrow cells transduced with control (MIP) or RE9a retrovirus and serially replated in methylcellulose. Data shown are averages with standard deviations of a representative dataset. Four independent assays were performed. (B) Typical colony images after 9^th replating from (A) taken using Nikon Eclipse TS100 microscope with 2×/0.06 objective lens and Nikon DS Camera Control Unit DS-U2 system. (C) Flow cytometric analysis of replated cells from (A). Cells from 3^rd, 6^th and 9^th replatings were stained for myeloid lineage markers Gr-1 and CD11b. Representative data from four independent assays shown. (D) and (E) Lack of <i>Alox5</i> decreases colony formation potential of hematopoietic cells transduced with MLL-AF9 and PML-RARα. Wildtype or <i>Alox5</i>-/- bone marrow cells were transduced with MIP and MLL-AF9 (D) or PML-RARα (E) retrovirus and serially replated in methylcellusose. Data shown are averages and standard deviations of representative datasets. Three independent assays were performed.</p

Cooperation between RUNX1-ETO9a and Novel Transcriptional Partner KLF6 in Upregulation of <i>Alox5</i> in Acute Myeloid Leukemia

<div><p>Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene <i>Alox5</i>, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of <i>Alox5</i> expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα <i>in vitro</i>. However, <i>Alox5</i> is not essential for the induction of leukemia by RUNX1-ETO9a <i>in vivo</i>. Finally, we demonstrate that the upregulation of <i>Alox5</i> by RUNX1-ETO9a occurs via the C₂H₂ zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, <i>KLF6</i> is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.</p></div

<i>Alox5</i> regulation by RE9a and KLF6.

<p>(A) Coregulation of <i>Alox5</i> reporter by RE9a and KLF6. <i>Alox5</i> −507 to +146 reporter co-transfected with RE9a, KLF6 or both. Expression was normalized to <i>Renilla</i> luciferase and control (Ctrl) was set to 1. (B) Knockdown of endogenous <i>KLF6</i> via shRNA. K562 cells were transfected with control or one of two independent shRNAs targeting <i>KLF6</i> and analyzed by qRT-PCR for <i>KLF6</i> expression. Expression values were normalized to <i>GAPDH</i> and control transfected value was set to 1. Data show averages with standard deviations of 3 independent transfections. (C) Knockdown of <i>KLF6</i> impairs ability of RE9a to upregulate <i>Alox5</i> promoter-luciferase reporter. K562 cells pre-transfected with control or <i>KLF6</i> shRNA were co-transfected with <i>Alox5</i> −507 to +146 reporter and RE9a. Expression was normalized to <i>Renilla</i> luciferase and control +RE9a was set to 100. (D) Mutation of KLF6 binding site significantly decreases activation of <i>Alox5</i> promoter by RE9a and KLF6. Wildtype or KLF6 binding site (b.s.)-mutated (GGGTG to GATCG) <i>Alox5</i> −30 to +146 reporter co-transfected with RE9a or KLF6. Expression was normalized to <i>Renilla</i> luciferase. Control (Ctrl) was set to 1. <i>p</i>-values are compared to wildtype reporter co-transfected with corresponding transgene. (E) KLF6 can interact with RUNX1, RE and RE9a. KLF6 and RUNX1, RE or RE9a were co-transfected into K562 cells, and lysates were immunoprecipitated with control or KLF6 antibody. Also shown is interaction with endogenous SP1. α-tubulin serves as a loading control for the whole cell lysate. After IP, KLF6 appears as multiple bands likely because KLF6 is expressed endogenously as multiple splicing isoforms which are enriched to more easily detectable levels by immunoprecipitation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003765#pgen.1003765-Narla1" target="_blank">[55]</a>.</p

Loss of <i>Alox5</i> does not block RE9a leukemia induction <i>in vivo</i>.

<p>(A) Survival of mice receiving wildtype or <i>Alox5</i>-/- fetal liver cells transduced by control (MigR1) or RE9a retrovirus. Number of mice in each cohort shown at right. WT median survival: 30.71 weeks; <i>Alox5</i>-/- median survival: 29.43 weeks; <i>p</i> = 0.39. (B) Presence of hematopoietic blast cells in tissues of mice transplanted with RE9a-transduced wildtype or <i>Alox5</i>-/- cells. Peripheral blood smears and cytocentrifugation of bone marrow and spleen cells were stained with Wright-Giemsa solutions. (C) Immunophenotype of myeloid progenitor cells in wildtype and <i>Alox5</i>-/- leukemias. Distribution of EGFP⁺Lin⁻Sca-1⁻c-Kit⁺ leukemic cells harvested from spleen shown based on expression of CD34 and Fcγ receptors II/III (FcγRII/III). At least 4 mice analyzed per genotype, with representative distributions shown.</p

Regulation of <i>KLF6</i> by RUNX1-ETO and RE9a.

<p>(A) Expression of <i>KLF6</i> in control- (Ctrl) or RE9a-transfected K562 cells. RE9a or empty vector was co-transfected into K562 cells along with a GFP-expressing vector to determine transfection efficiency. <i>KLF6</i> mRNA levels were normalized to <i>GAPDH</i> with Ctrl set to 1, and samples were then normalized to account for transfection rate by percent GFP-expressing cells as determined by flow cytometry. Data show averages and standard deviations of three independent transfections. (B) Expression of <i>KLF6</i> in HL60 [t(8;21)-negative] and SKNO and Kasumi-1 [t(8;21)-positive] cell lines. <i>KLF6</i> mRNA levels were normalized to <i>GAPDH</i> and HL60 was set to 1. Data show averages and standard deviations of 3 independent RNA isolations. (C) Expression of <i>KLF6</i> in control-, RUNX1-ETO- or RUNX1-ETO9a-transduced HL60 cells. Following 2 rounds of retroviral transduction, <i>KLF6</i> levels determined as in (B), with control-transduced cells (Ctrl) set to 1. Data display averages and standard deviations of 3 independent transductions.</p

Upregulation of <i>Alox5</i> in acute myeloid leukemia and by RUNX1-ETO9a.

<p>(A) Normalized log₂ expression of <i>Alox5</i> in control or RE9a-leukemic murine lin⁻c-Kit⁺ bone marrow cells. mRNA transcript levels were normalized to <i>Gapdh</i> and control was set to 1. Data show averages and standard deviations from 3 independent mice each. (B) RE9a regulation of mouse <i>Alox5</i> promoter-luciferase reporter. Numbers indicate base pair relative to transcription start site. Two RUNX1 binding sites (TGTGGT) were either wildtype or mutated to TGTtag to abrogate RE9a binding <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003765#pgen.1003765-Meyers1" target="_blank">[54]</a>. Indicated promoter-luciferase reporter was co-transfected with control (Ctrl) or RE9a plasmid and expression was normalized to <i>Renilla</i> luciferase. Wildtype promoter+control set to 1. (C) RE9a regulation of truncated mouse <i>Alox5</i> promoter-luciferase reporter. Luciferase assay performed as described in (B), with −1783 to +146 promoter+control set to 1.</p

Analysis of <i>Alox5</i> promoter regulation by RE9a.

<p>(A) Schematic of <i>Alox5</i> promoter-luciferase reporter with motifs differing from RUNX1 consensus binding site (TGYGGT) indicated. Bases differing from consensus site labeled in lowercase. Transcription factor SP1 binding site also indicated. Numbers represent base pairs relative to transcription start site. (B) Basal regulation of <i>Alox5</i> promoter-luciferase truncations. Indicated reporters were transfected in the absence of RE9a and expression was normalized to <i>Renilla</i> luciferase and the −507 to +146 construct was set to 1. * = <i>p</i><0.01 relative to −507 reporter. (C) Inducible regulation of <i>Alox5</i> promoter-luciferase by RE9a. Indicated reporters were co-transfected with control or RE9a and expression was normalized to <i>Renilla</i> luciferase. Each control (Ctrl) transfection normalized to 1. <i>p</i>-value relative to −507 reporter +RE9a.</p

Coexpression of USP18 increases electrogenic peptide transport in PEPT2-expressing <i>Xenopus laevis</i> oocytes.

<p><b>A:</b> Representative original tracings showing I_gly-gly in <i>Xenopus laevis</i> oocytes injected with water (a) or expressing USP18 alone (b) or expressing PEPT2 without (c) or with additional coexpression of wild type USP18 (d). <b>B:</b> Arithmetic means ± SEM (n = 8–9) of I_gly-gly in <i>Xenopus</i> oocytes injected with water (striped bar), expressing USP18 alone (grey bar), or expressing PEPT2 without (white bar) or with (black bar) wild type USP18. **(p<0.01) indicates statistically significant difference from the absence of USP18.</p

<i>ISG15</i> expression during erythroid differentiation is independent of IFN signaling and partially dependent upon Epo signaling.

<p>(<b>A</b>) semi-quantitative RT-PCR analyses of <i>ISG15, Ube1L, UbcM8, Herc6, Irf7, Bcl-x_L</i> and <i>ß-Major-Globin</i> mRNA expression in WT or <i>IFNAR^-/-</i> differentiating primary erythroblasts. Bone marrow erythroblasts of each genotype were maintained in proliferation conditions (SCF, Epo and Dex) for one week and next induced to differentiate in response to Epo alone for three days. Cells were collected every 24 hours as indicated and RNA extracted. The experiment was normalized to <i>ß-Actin</i> mRNA expression. (<b>B</b>) Statistical analysis of the induction of the expression of <i>ß-Maj-Globin, Bcl-X_L</i>, <i>ISG15</i>, <i>Ube1L</i>, <i>UbcM8</i> and <i>Herc6</i> mRNA in differentiating WT and IFNAR^-/- erythroblasts. Quantification was performed as described in Mat & Met. Note the unchanged expression of a second housekeeping gene <i>HPRT</i>. au = arbitrary unit. (<b>C</b>) Whole cell protein extracts were prepared from WT or <i>IFNAR^-/-</i> erythroblasts maintained as in A and analyzed on a 10% acrylamide gel for <i>ISG15</i> expression using an anti-ISG15 antibody. Anti-ß-Actin and anti-GAPDH were used as loading controls. (<b>D</b>) Statistical analysis of the induction of ISG15 during erythroid differentiation as normalized to ß-Actin. Quantification was performed as described in Mat & Met. au = arbitrary unit. (<b>E and F</b>) A p53^-/- erythroid cell line expressing exogenous hBcl-X_L was switched from proliferation conditions (Epo, SCF, Dex) to differentiating medium in the presence or absence of Epo. Cells were collected every 24 hours as indicated and analyzed for their ability to (e) survive as measured by propidium iodide staining in flow cytometry analyses; (f) differentiate as shown by analysis of their morphology after benzidine/May-Grunwald staining. Note the significant induction of cell death in control cells maintained in absence of Epo; in contrast, hBCL-xL erythroblasts are strongly protected from apoptosis. (<b>G</b>) Cells were lyzed according to cell number and volume. ISG15 expression was analyzed on a 15% acrylamide gel using anti-ISG15 antibody, activation of the EpoR/STAT5 signaling pathway was monitored using anti-P-STAT5 antibody, differentiation was monitored using anti-Globin antibody and loading control was performed using anti-SAM68 antibody. (<b>H</b>) Mock and mscv-puro-STAT5^S710F transduced p53^-/- erythroid cell line maintained under proliferation conditions were lyzed and analyzed for ISG15 expression using anti-ISG15 antibody on a 10% acrylamide gel (Top panel). P-STAT5 was detected at a higher level in mscv-puro-STAT5^S710F transduced cells while only a modest increase in the total amount of STAT5 can be noted. Anti-β -Actin was used as a loading control.</p

Coexpression of Nedd4-2 decreases electrogenic peptide transport in PEPT1-expressing <i>Xenopus laevis</i> oocytes.

<p><b>A:</b> Representative original tracings showing I_gly-gly in <i>Xenopus laevis</i> oocytes injected with water (a), expressing PEPT1 alone (b) or with USP18 (c), Nedd4-2 (d), or with USP18 and Nedd4-2 (e). <b>B:</b> Arithmetic means ± SEM (n = 13–15) of I_gly-gly in <i>Xenopus laevis</i> oocytes injected with water (striped bar) or expressing PEPT1 without (white bar) or with USP18 (black bar), with Nedd4-2 (light grey bar), or with USP18 and Nedd4-2 (dark grey bar). *(p<0.05) indicates statistically significant difference from oocytes expressing PEPT1 alone.</p