Downregulation of rRNA Transcription Triggers Cell Differentiation

<div><p>Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.</p></div

TIF-IA KD-induced cell differentiation of mouse HSCs in ex vivo culture.

<p>(A) Scheme showing the experimental procedure used for the HSC ex vivo culture system. The HSCs were purified from 8- to 12-week-old wild type mice. The purified HSCs were transduced with a lentivirus that expressed a shRNA against TIF-IA and cultured in media containing SCF and TPO. On days 5, 7, 10, and 12 after lentivirus transduction, myeloid differentiation was analyzed by flow cytometry. (B, C) TIF-IA KD promoted the myeloid differentiation of HSCs in culture. Anti-Mac-1 and anti-Gr-1 were used as myeloid cell markers. (B) The upper and lower panels show the results for the shControl and shTIF-IA cultures, respectively. Each panel shows the flow cytometric profiles of GFP⁺ transduced cells. (C) The percentages of lineage⁻ cells (Mac-1⁻Gr-1⁻) and myeloid cells (Mac1⁺ single positive and Mac-1⁺Gr-1⁺) among the GFP⁺ cells are shown as bar graphs. Values are expressed as the mean ± S.D., <i>n</i> = 3. *<i>P</i><0.05.</p

Suppression of rRNA transcription by actinomycin D (Act D) induced the differentiation of HL-60 and THP-1 cells.

<p>(A) A low concentration of Act D inhibited rRNA transcription in HL-60 and THP-1 cells. HL-60 and THP-1 cells were treated with 5 nM Act D for 24 h. The levels of pre-rRNA were determined by real-time quantitative PCR (RT-qPCR) and normalized by cell number. (B) Act D induced the expression of CD11b in HL-60 and THP-1 cells. Cells were cultured in the absence (control) or presence of all-trans-retinoic acid (ATRA) (1 µM), PMA (10 ng/mL), or Act D (5 nM) at 37°C. After 3 days, the CD11b expression levels were determined by flow cytometry (left panels). The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). (C, D) Inhibition of the cell cycle did not affect CD11b expression. THP-1 cells were treated with PMA (10 ng/mL), Act D (5 nM), or roscovitine (15 µM) for 3 days. (C) The DNA content was determined by DAPI and analyzed by flow cytometry. Similar results were obtained in three independent experiments. (D) The CD 11b expression levels were determined by flow cytometry. The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). (E) Roscovitine treatment did not affect the pre-rRNA levels. THP-1 cells were treated with PMA (10 ng/mL), Act D (5 nM), or roscovitine (15 µM) for 3 days. The pre-rRNA levels were determined by RT-qPCR and normalized by cell number. Values are expressed as the mean ± S.D., <i>n</i> = 3. *<i>P</i><0.05. N.S.: <i>P</i>>0.05.</p

Suppression of rRNA transcription by TIF-IA KD induced the differentiation of HL-60.

<p>(A, B) siRNA-TIF-IA reduced the mRNA and protein levels of TIF-IA. HL-60 cells were transfected with siRNAs for luciferase (siCont.) and TIF-IA (siTIF-IA#1 or siTIF-IA#2), and cultured for 3 days. (A) The mRNA levels of TIF-IA were determined by RT-qPCR. (B) The protein levels of TIF-IA were determined by immunoblotting. (C) The pre-rRNA levels were determined by RT-qPCR. (D, E) TIF-IA KD induced the differentiation of HL-60 cells. (D) CD11b expression was determined by flow cytometry. ATRA (1 µM) was used as the positive control. The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). We present the same histograms for siCont. and ATRA in the upper and lower panels because these experiments were performed at the same time. (E) The MPO levels were determined by RT-qPCR and normalized by the cyclophilin levels. Values are expressed as the mean ± S.D., <i>n</i> = 3 (A, B, D). Values are expressed as the mean ± S.D., <i>n</i> = 4 (C). *<i>P</i><0.05.</p

(A) Enforced expression of Noxa-induced cell death in effector Th2 cells after cytokine depletion

Effector Th2 cells infected with a –containing retrovirus were cultured in vitro for 24 h without cytokines. hNGFR profiles (left) and annexin V staining profiles of the electronically gated hNGFR (gate #2) and hNGFR (gate #1) populations are shown. Three independent experiments were performed with similar results. (B) KJ1 effector Th2 cells infected with –containing retrovirus were transferred into BALB/c mice. 5 wk later, memory Th2 cell generation was determined by KJ1/EGFP expression. Expression of EGFP in pretransferred effector Th2 cells (top left) and a typical KJ1/GFP profile of freshly prepared memory Th2 cells (top right) are shown. In the bottom panels, the percentages of KJ1 cells and GFP Noxa-overexpressing cells and the mean fluorescence intensity of the GFP cells are shown with standard deviations ( = 4). The experiments were performed twice with similar results. (C) The effector Th2 cells from /, /, and / mice (Ly5.2) were transferred into Ly5.1 host mice, and the number of Ly5.2 memory Th2 cells was determined. A typical staining pattern of CD4/Ly5.2 (top) and the percentages of Ly5.2 cells among CD4 T cells are shown with standard deviations ( = 5; bottom). Three independent experiments were performed with similar results. (D) Deletion of the gene enhanced the generation of memory Th2 cells. In vitro–generated effector Th2 cells (Ly5.2) were transferred into Ly5.1 host mice. 5 wk after cell transfer, the number of Ly5.2 memory Th2 cells was determined. A representative CD4/Ly5.2 profile (left) and the mean values with standard deviations ( = 5; right) are shown. The experiments were performed twice with similar results.<p><b>Copyright information:</b></p><p>Taken from "Bmi1 regulates memory CD4 T cell survival via repression of the gene"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1109-1120.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373843.</p><p></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

Identification of BMI1 Promoter Inhibitors from <i>Beaumontia murtonii</i> and <i>Eugenia operculata</i>

B-Cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) is a core component of the polycomb repressive complex 1 (PRC1). Abnormal expression of BMI1 is associated with a number of human malignances and cancer stem cells (CSCs), which cause chemotherapy resistance. Therefore, small molecules that inhibit BMI1 expression are potential candidates for cancer therapy. In this study, a cell-based reporter gene assay was developed that allowed BMI1 promoter activity to be measured in 293T human embryonic kidney cells based on luciferase expression levels. Using this screening assay, the methanol-soluble extracts of <i>Beaumontia murtonii</i> and <i>Eugenia operculata</i> were selected as leads. Bioassay-guided fractionation of the extracts led to the isolation of three known cardenolides (<b>1</b>–<b>3</b>) and one new compound (<b>4</b>) from <i>B. murtonii</i> and two known triterpenoids (<b>5</b> and <b>6</b>) and one new compound (<b>7</b>) from <i>E. operculata</i>. These seven compounds inhibited BMI1 promoter activity (IC₅₀ range 0.093–23.0 μM), and the most active compound, wallichoside (<b>1</b>), was further evaluated. Western blot analysis revealed that wallichoside (<b>1</b>) decreases BMI1 protein levels in HCT116 human colon carcinoma cells, and flow cytometry analysis showed that it significantly reduced levels of the CSC biomarker epithelial cell adhesion molecule. Wallichoside (<b>1</b>) also inhibited sphere formation of Huh7 human hepatocellular carcinoma cells, indicating that it diminished the self-renewal capability of CSCs

Generation of conditional knockout allele for <i>Pcgf5</i> in mice.

<p>(A) RT-PCR analysis of <i>Pcgf5</i> in BM hematopoietic cell fractions. Cells analyzed were CD34^-LSK long-term HSCs, CD34⁺Flt3^-LSK short-term HSCs, CD34⁺Flt3⁺LSK multipotent progenitors (MPPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and lineage marker⁺ mature hematopoietic cells. <i>Hypoxanthine-guanosine phosphoribosyl transferase (Hprt)</i> was used as a housekeeping control gene. Data are shown as the mean ± standard deviation (SD) for triplicate analyses. (B) Strategy for making a conditional knockout allele for <i>Pcgf5</i> by homologous recombination in ES cells. FRT recombinase was used to remove the <i>Neo</i> cassette. (C) Scheme of the hematopoietic repopulation assay. Total BM cells (5x10⁶ cells) from <i>Cre-ERT</i> and <i>Cre-ERT;Pcgf5</i>^<i>fl/fl</i> were transplanted into lethally irradiated CD45.1 recipient mice without competitor BM cells, or 2x10⁶ total BM cells were transplanted with the same number of competitor BM cells. To induce deletion of <i>Pcgf5</i>, 100 μl of tamoxifen (10 mg/ml) was intraperitoneally injected once a day for five consecutive days at 1 month post-transplantation. (D) Efficient deletion of <i>Pcgf5</i> in hematopoietic cells detected by genomic PCR. Deletion of <i>Pcgf5</i> in <i>Cre-ERT;Pcgf5</i>^<i>fl/fl</i> PB myeloid cells was evaluated pre- and post-tamoxifen treatment. “WT”, “Floxed”, and “<i>Δ</i>” alleles indicate the wild-type and floxed <i>Pcgf5</i> allele, and floxed <i>Pcgf5</i> allele after removal of exon 2 by Cre recombinase, respectively. (E) Detection of truncated <i>Pcgf5</i> mRNA in BM <i>Pcgf5</i>^<i>Δ/Δ</i> LK cells using primers directed to exon 1 and exon 5/6 junction. (F) Pcgf5 interacts with Ring1B. Ring1B in lysates from <i>Pcgf5</i>^<i>fl/fl</i> and <i>Pcgf5</i>^<i>Δ/Δ</i> ES cells was immunoprecipitated using anti-Ring1b antibody, and then immunoprecipitates were detected by immunoblotting using anti-Ring1b and anti-Pcgf5 antibodies.</p

Depletion of <i>Pcgf5</i> does not compromise adult hematopoiesis.

<p>(A) PB cell counts of recipients repopulated with <i>Cre-ERT</i> (+/+) and <i>Cre-ERT;Pcgf5</i>^<i>fl/fl</i> BM cells after deletion of <i>Pcgf5</i> (<i>Δ/Δ</i>) by tamoxifen injection. Data are shown as mean ± SD (n = 4–5). (B) Lineage contribution of donor cells to myeloid (Gr-1⁺ and/or Mac-1⁺), B (B220⁺), or T (CD4⁺ and/or CD8⁺) cells in the PB shown as mean ± SD (n = 4–5). (C) Absolute number of CD45.2⁺ donor-derived hematopoietic cells in a unilateral pair of femur and tibia of recipients at 5 months post-transplantation. Data are shown as mean ± SD (WT, n = 5; <i>Pcgf5</i>^<i>Δ/Δ</i>, n = 6). (D) Absolute number of CD45.2⁺ donor-derived LSK cells, CLPs and myeloid progenitors in the BM of recipient mice at 5 months post-transplantation presented as mean ± SD (WT, n = 5; <i>Pcgf5</i>^<i>Δ/Δ</i>, n = 6). n.s., not significant.</p

(A) mRNA expression of and p53-related proapoptotic genes in effector Th2 cells was determined by quantitative RT-PCR

The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. Three independent experiments were performed with similar results. (B) mRNA expression of , p53-related proapoptotic genes, and antiapoptotic genes in memory Th2 cells was analyzed. The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. Two independent experiments were performed with similar results. (C) Effects on p16 and p19 deficiency on the memory Th2 cell generation. The effector Th2 cells from the indicated mice (Ly5.2 background) were transferred into Ly5.1 host mice. 5 wk after cell transfer, the number of Ly5.2 memory Th2 cells was determined. The mean values are shown with standard deviations ( = 5; right). The experiments were performed twice with similar results. (D) mRNA levels of proapoptotic genes in // effector Th2 cells were determined by quantitative RT-PCR. The relative intensity (/HPRT; mean of three samples) is shown with standard deviations. The experiments were performed twice with similar results.<p><b>Copyright information:</b></p><p>Taken from "Bmi1 regulates memory CD4 T cell survival via repression of the gene"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1109-1120.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373843.</p><p></p