Variant rs1801157 of the SDF-1ß 3’UTR, miRNA941 and G-CSF responsiveness of healthy stem cell donors.

<p><b>(A)</b> Sequence alignment of miR941 with WT- and SNP-variants of the 3’UTR of CXCL12. (<b>B)</b> Representative results from the sequence-specific PCR established to distinguish between WT- and SNP-variant. (<b>C)</b> Representative sequencing results from the 3’UTR genotyping. (<b>D)</b> Expected and observed frequencies of WT, heterozygous and homozygous SNP genotypes. (<b>E)</b> Circulating CD34+ cells after 9 doses q12h of G-CSF by CXCL12 3’UTR genotype. Mobilization efficiency for WT, heterozygous SNP and homozygous SNP donors was 94.9±2.9, 101.6±4.3 and 88.0±10.0 CD34+ cells/μl (mean±SEM), respectively. All donors combined are shown in dark grey bars, separate analyses by gender are overlaid. (<b>F)</b> miRNA expression in non-hematopoietic BM cells from healthy volunteer donors was tested by global miR expression arrays; individual highly and lowly expressed miRNAs were further tested by real-time PCR. miR941 expression was barely detectable. (<b>G)</b> The interaction between miR941 and the 3’UTR was assessed by dual luciferase assays. Luciferase activity was the same for the WT- and SNP-variant of the CXCL12 3’UTR, in the presence or absence of WT or mutated miR941. Hsa-miR1255 served as positive control for miRNA-mediated down-regulation of luciferase activity (not shown).</p

Custom oligonucleotides used for these studies.

<p>PCR conditions are available from the authors upon request.</p

Betaine feeding improves hFVIII and hFIX secretion <i>in vivo</i>.

<p>(A–F) 24 hours post minicircle FVIII-BDD gene transfer the FVIII knockout mice received water without (group I) or with 2% betaine supplementation in the drinking water (group II, each n = 10). After 3 days plasma samples were collected and each group was monitored for human FVIII antigen (A and B) and related activity levels (D and E) and group treatment was switched. After another 3 days, plasma levels were tested again. (C and F) represent the calculated overall effect of betaine on FVIII antigen levels (C) or FVIII activity (D). square symbols indicate samples of the first measuring point, triangles the second one. (G–I) After reaching stable FIX expression levels following minicircle FIX gene transfer, FIX knockout mice were fed 2% Betaine-supplemented drinking water ad libitum in a crossover-study of two groups. After 3 and 17 days of treatment, retroorbitally collected plasma samples were monitored for human FIX antigen levels (G and H). (I) shows the overall change in FIX expression from both groups after 17 days of administration. All values are represented as mean ± SEM. Same symbols indicate samples of the same mouse at different time points; clear: tap water treatment (control), filled: betaine administration. Student’s t-test ((G) ANOVA). *<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p

CC improve secretion of FVIII-BDD, FVIII-BDD-eGFP and FVIII-FL <i>in vitro</i>.

<p>Heterologous CHO cells were incubated with CC at different concentrations. FVIII activity was determined in cell supernatants after 72 h by chromogenic assay. (A) Effect of the following CC on human (h)FVIII-BDD secretion: Betaine (100; 50; 25 mM), ectoine (150; 100; 50 mM), trehalose (150; 100; 50 mM), sorbitol (150; 100; 50 mM), taurine (150; 100; 50 mM), trimethylamine N-oxide (TMAO;50; 25; 12,5 mM) and sodium 4-phenylbutyrate (4-PBA; 2; 0,4 mM). Number of experiments, n = 2. (B) Effect of betaine, ectoine, and the endoplasmatic ATPase inhibitors curcumin and thapsigargin on FVIII-.BDD-eGFP secretion. Butylated hydroxyanisole (BHA) is added as treatment control. n = 3. The mean FVIII secretion level ± SD of untreated hFVIII-BDD-eGFP expressing cells was 19±12 IU per 10e6 cells per 72 h. (C) FVIII-BDD-eGFP secretion into cell supernatants over time at different betaine concentrations. n = 3. (D) Influence of betaine, ectoine, curcumin and thapsigargin on FVIII-FL secretion 72 hours following drug supplementation. n = 3. All values are presented as means ± SEM. ANOVA test * <i>P</i><.05; ** <i>P</i><.001.</p

Rescue of mutant FVIII proteins <i>in vitro</i> and <i>in vivo</i>.

<p>(A–D) HepG2 cells expressing hFVIII muteins were incubated with CC for 72 h. Amount of hFVIII activity in cell supernatant of HepG2 cells expressing hFVIII-BDD Q305P (A and C) or hFVIII-BDD W2313A (B and C) was measured 72 h post betaine treatment. (A and B) show the supplementation of single CC and (C) betaine-ectoine combined treatment. (D) Post CC incubation HepG2 hFVIII-BDDQ305P cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. hFVIII antigen was determined in both fractions by indirect ELISA. (E–J) hFVIII-BDDQ305P injected Hem A mice were treated with 2% betaine ad libitum per os in a crossover-study of two groups (each n = 10). After 3 days of treatment hFVIII antigen and activity was measured and treatment was switched between mouse groups. 3 days later, plasma levels were tested again. (E and F) show hFVIII antigen levels, (H and I) the related hFVIII activity levels in plasma of group I or II. (G and J) represent the calculated overall effect of betaine on FVIII antigen levels (G) or FVIII activity (J). square symbols indicate samples of the first measuring point, triangles the second one; clear: tap water-administration (control), filled: 2% betaine administration. (K and L) Endogenous murine FIX levels in all injected FVIII knockout mice (K) and murine FVIII levels of all used FIX knockout mice (L) with and without betaine in the drinking water. Normal mouse levels were set to 100%. Values are presented as means ± SEM. (A–D) ANOVA; (E–H; K–L) Student’s t-test; (I–J) Wilcoxon signed rank test;*<i>P</i><.05, **<i>P</i><.005, ***<i>P</i><.0005.</p

Betaine increases solubility of intracellular FVIII.

<p>CHO cells expressing eGFP-tagged FVIII-BDD protein were incubated with and without betaine. (A,B) Flow cytometry analysis was used to determine the eGFP-signal in untreated cells versus cells treated with betaine or control substance BHA. The mean eGFP intensity (X mean GFP) in the range M1 was used as distinctive parameter. (A) representative histogram after 48 h of treatment and (B) values after 72 h presented as means ± SEM of 3 independent experiments. ANOVA **<i>P</i><.001. (C,D) After 72 h incubation cells were successively lysed in PBS/0.5% Triton X-100 and PBS/1% SDS. (C) FVIII antigen was determined in both fractions by indirect ELISA. (D) Triton X-100-soluble and insoluble fractions were separated on SDS-polyacrylamid gradient gels, and hFVIII light chains (lc), eGFP in hFVIII-single chain (sc) and GAPDH were detected by Western blot. Δ indicates lower band of hFVIII lc doublet.</p

Inhibition of RUNX1/ETO increases miR144/451 expression.

<p><b>(A)</b> Expression of endogenous RUNX1/ETO in Kasumi1, SKNO1 and two RUNX/ETO positive patient samples. Expression of RUNX1/ETO in Kasumi1 cells was set as 1 and the expression levels of the SKNO1 cells and two patient samples are shown as fold compared to Kasumi1 cells. HEK293 and hCD34+ serve as RUNX1/ETO negative controls. Values were gathered by q-RT-PCR and normalised to GAPDH. <b>(B)</b> Expression of miR144/451 in Kasumi1, SKNO1 and two different patient samples. HEK293 and hCD34+ served as miR144/451 negative and positive controls, respectively. Q-RT-PCR values were normalised to GAPDH expression and are shown as fold relative to values gathered with Kasumi1 cells. <b>(C)</b> Knock-down of RUNX1/ETO in Kasumi1 cells. Knock-down of RUNX1/ETO by an shRNA targeting the R/E fusion site leads to increased miR144/451 expression. <b>(D)</b> Treatment of Kasumi1 cells with trichostatin-A (TSA) leads to degradation of RUNX1/ETO. Kasumi1 cells were treated with the indicated concentrations of TSA for 24 hours. Protein expression was determined by Western blot using an anti-ETO antibody. R/E runs at about 100 kD. As loading control (l.c.) a protein band running at 55 kD visible upon Ponceau S staining of the membrane is shown. <b>(E)</b> MiR144/451 expression is up-regulated in Kasumi1 cells upon TSA treatment. MiR144/451 expression upon treatment with TSA was measured by q-RT-PCR. Values were normalised to GAPDH expression. <b>(F)</b> MiR144/451 expression is up-regulated in patient samples upon TSA treatment. Cells were treated with 1 uM TSA for 24 hours. Q-RT-PCR values were normalised to GAPDH expression. Q-RT-PCR against RUNX/ETO was performed with specific primers detecting the fusion protein. Error bars give the standard deviation from at least four independent determinations. The P-values were calculated using Student’s t test. **P <0.01, ***P <0.001.</p

Regulation of miR144/451 expression.

<p><b>(A)</b> Schematic representation of the miR144/451 locus. The first 4700 bp of the 5’-region of miR144/451 are shown. Binding sites for hematopoietic transcription factors are found about 4500 bp upstream (enhancer) and in the proximal promoter region (promoter). A less conserved region (not conserved, n.c.) separates these regions. <b>(B)</b> Luciferase reporter gene experiment with constructs harbouring the enhancer or the promoter region of the miR144/451 cluster. Reporter constructs were transfected into HEK293 cells or K562 cells, respectively. Relative light units (RLU) are given as fold induction compared to values gathered with empty luciferase vector. Luciferase values were normalised for transfection variations by measuring the activity of a cotransfected beta-galactosidase expression vector. Error bars indicate the standard deviation from six independent transfections and measurements. <b>(C)</b> ChIP assay in primary hCD34+ cells shows binding of TAL1 predominantly to the enhancer region (enh.) as opposed to the promoter region (prom.). <b>(D)</b> ChIP assay in hCD34+ cells shows binding of RUNX1 to the promoter region (prom.) but only little to the enhancer region (enh.). <b>(E)</b> The human miR144/451 promoter contains a conserved binding site for RUNX1. The RUNX1 binding site was mutated by changing two base pairs in the core RUNX1 site. <b>(F)</b> Luciferase reporter assay using the wild type (wt prom) and the mutated (RUNXmut) miR144/451 promoters, respectively. Transfection of RUNX1, the RUNX1/ETO (R/E) fusion protein or the truncated RUNX1/ETO (R/Etr) repressed wild type (wt prom), but not the mutated promoter (RUNX1mut prom). Values are presented as fold change compared to the relative light units gathered upon transfection of the reporter gene and empty expression vector. Luciferase values were normalised for transfection variations by measuring the activity of a cotransfected beta-galactosidase expression vector. Error bars represent the standard deviation from six independent transfections and measurements. The P-value was calculated using Student’s t test. **P <0.01.</p

MiR144/451 expression is epigenetically repressed during megakaryocytic differentiation.

<p><b>(A)</b> MiR144/451 expression is decreased upon megakaryocytic differentiation and increased upon erythroid expression of primary hCD34+ cells. Differentiation was done for 6 days (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005946#pgen.1005946.s007" target="_blank">S7 Fig</a>). Expression of the endogenous pri-microRNA was measured by q-RT-PCR. <b>(B)</b> ChIP assay analysis of RUNX1 binding to the miR144/451 promoter during erythroid (CD34-E) or megakaryocytic (CD34-M) differentiation of hCD34+ (CD34) cells. <b>(C)</b> Analysis of RUNX1 protein abundance in hCD34+ cells and upon megakaryocytic differentiation (CD34-M) by Western blot using a RUNX1 antibody and a lamin antibody as loading control. <b>(D-I)</b> Cofactor and histone modification changes at the promoter of miR144/451 during megakaryocytic differentiation (CD34-M) using ChIP analysis in hCD34+ cells. <b>(D-F)</b> ChIP analysis reveals altered binding of PRMT6, p300 and WDR5 to the miR144/451 promoter upon megakaryocytic differentiation. <b>(G-I)</b> ChIP reveals altered H3R2me2, H3K9ac and H3K4me3 at the miR144/451 promoter upon megakaryocytic differentiation. <b>(D-I)</b> Q-PCR values are given as percent input. Histone modification ChIP values were corrected by a Histone 3 ChIP for nucleosome density. The error bars represent the standard deviation from at least four independent determinations. The P-values were calculated using Student’s t test. **P <0.01, ***P <0.001.</p

Identification of RUNX1 regulated microRNAs.

<p><b>(A)</b> Schematic representation of the experimental setup. K562 cells were transduced with RUNX1 expression vector or empty vector control. The transductions were performed in independent triplicates and differentially expressed microRNAs were determined by small-RNA sequencing. <b>(B)</b> 588 small RNAs were differentially expressed upon RUNX1 expression in K562 cells. 31 were snRNAs (small nuclear RNA), 45 rRNAs (ribosomal RNA), 142 snoRNAs (small nucleolar RNA) and 370 microRNAs. RNAs were included if they displayed an at least -0.5 or +0.5 log2-fold change and a P-value <0.05. <b>(C)</b> Of the 370 identified microRNAs, 237 were up-regulated and 133 down-regulated upon RUNX1 expression. <b>(D)</b> Schematic representation of hematopoiesis from the stem cell throughout the myeloid lineage. Those microRNAs are shown, which were altered upon RUNX1 over-expression. The green arrow indicates a positive role and the red blunted arrow indicates a negative role in differentiation according to published work. HSC: hematopoietic stem cell, MPP: multipotent progenitor, CMP: common myeloid progenitor, GMP: granulocyte monocyte progenitor, MEP: megakaryocyte erythrocyte progenitor, MkP: megakaryocyte progenitor, EP: erythrocyte progenitor. <b>(E)</b> Independent evaluation of microRNA expression. A subset of mature microRNAs influenced by RUNX1 and with a role in myeloid differentiation identified by RNA-sequencing, was tested by q-RT-PCR. Q-RT-PCR values are given as relative expression of RUNX1 transduced K562 cells, compared to K562 cells transduced with empty vector. Values were normalised to RNU6-2 expression. The error bars give the standard deviation from four independent determinations. All values were significantly different from the control according to Student’s t test P <0.05. <b>(F)</b> RUNX1 over-expression leads to a decrease of miR144/451 (pri-microRNA) transcript. Q-RT-PCR values are shown as fold expression compared to empty vector transduced K562 cells. Error bars represent the standard deviation from four independent determinations. The P-value was calculated using Student’s t test. **P <0.01.</p