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

Requirement of Aurora B in the Cdc2-independent chromosomal targeting of condensin I

<p><b>Copyright information:</b></p><p>Taken from "Analysis of the role of Aurora B on the chromosomal targeting of condensin I"</p><p></p><p>Nucleic Acids Research 2007;35(7):2403-2412.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874644.</p><p>© 2007 The Author(s)</p> Interphase extract was depleted with anti-Aurora B (lane 7), anti-Aurora A (lane 8) or anti-Cdc2 antibodies (lane 9). As a standard, 100% (lane 1) of mitotic extract, 100% (lane 2), 50% (lane 3), 25% (lane 4), 12.5% (lane 5) and 6.25% (lane 6) of interphase extract were loaded in parallel. Efficiency of immunodepletion was measured by quantitative immunoblotting using the antibodies indicated. Sperm nuclei were incubated with mitotic extract (lanes 1–4), or interphase extract (lane 5), OA-treated interphase extract (lane 6), OA-treated interphase extract depleted of Aurora B (lane 7), OA-treated interphase extract depleted of Aurora A (lane 8), and OA-treated interphase extract depleted of Cdc2 (lane 9). Chromatin or chromosomes were isolated, and 100% (lanes 1, 5–9), 50% (lane 2), 25% (lane 3) and 12.5% (lane 4) of the samples were blotted by anti-phospho-H3 antibody (upper), anti-XCAP-C (middle) or anti-XCAP-E (lower) antibodies. In the case of blotting with anti-condensin subunit antibodies, a lower amount of samples was used as a standard, namely, 25% (lane 1), 12.5% (lane 2), 6.3% (lane 3) and 3.1% (lane 4). Sperm chromatin was incubated with mitotic extract (a), interphase extract (b), OA-treated interphase extract (c), OA-treated interphase extract depleted of Aurora B (d), OA-treated interphase extract depleted of Aurora A (e) and OA-treated interphase extract depleted of Cdc2 (f). Samples were fixed and stained with Hoechst

Requirement of condensin in OA-dependent partial chromosome condensation in the interphase extract

<p><b>Copyright information:</b></p><p>Taken from "Analysis of the role of Aurora B on the chromosomal targeting of condensin I"</p><p></p><p>Nucleic Acids Research 2007;35(7):2403-2412.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874644.</p><p>© 2007 The Author(s)</p> Mitotic (lanes 1–2) and interphase (lanes 3–4) extracts were mock-depleted (lanes 1 and 3) or depleted with a mixture of affinity-purified condensin antibodies (lanes 2 and 4). Equal volumes of these extracts were subjected to SDS-PAGE, blotted, and detected using the indicated antibodies. Sperm chromatin was incubated with mock-depleted mitotic extract (lanes 1–4), condensin-depleted mitotic extract (lane 5), mock-depleted interphase extract (lane 6), mock-depleted interphase extract supplemented with OA (lane 7), condensin-depleted interphase extract (lane 8) or condensin-depleted interphase extract supplemented with OA (lane 9) at 22°C for 2 h. The assembled structures were isolated, and 25 (lane 1), 12.5 (lane 2), 6.3 (lane 3), 3.1 (lane 4) and 100% (lanes 5–9) were analyzed by immunoblotting with the indicated antibodies. Sperm chromatin was incubated with mitotic extract (a), condensin-depleted mitotic extract (b), mock-depleted interphase extract (c), mock-depleted interphase extract with OA (d), condensin-depleted interphase extract (e), or condensin-depleted interphase extract with OA (f) at 22°C for 2 h. After incubation, assembled chromatin structures were fixed and stained with Hoechst. Bar, 10 μm

Stimulation of chromosomal binding of condensin I by OA in interphase extracts

<p><b>Copyright information:</b></p><p>Taken from "Analysis of the role of Aurora B on the chromosomal targeting of condensin I"</p><p></p><p>Nucleic Acids Research 2007;35(7):2403-2412.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874644.</p><p>© 2007 The Author(s)</p> Sperm nuclei were incubated with mitotic extract (lanes 1–3), interphase extract (lanes 4–6), interphase extract supplemented with 1.2 (lanes 7–9), 3.6 (lanes 10–12) or 12 μM (lanes 13–15) of OA at 22°C for 2 h. Chromatin-bound proteins were dissolved with SDS-PAGE sample buffer, and 12.5 (lanes 1, 4, 7, 10 and 13), 25 (lanes 2, 5, 8, 11 and 14) and 50% (lanes 3, 6, 9, 12 and 15) of each sample were separated by SDS–PAGE, and immunoblotted with anti-phospho H3. Samples were prepared as described in (A), and 6.25% (lane 1), 12.5% (lanes 2, 4, 7, 10 and 13), 25% (lanes 3, 5, 8, 11 and 14) and 50% (lanes 6, 9, 12 and 15) of samples were blotted using anti-XCAP-E (upper), and anti-XCAP-G (lower) antibodies. Sperm chromatin was assembled in the mitotic extract (a, d), interphase extract (b, e, g), and interphase extract supplemented with 3.6 μM OA (c, f, h). Samples were fixed and stained with Hoechst (a, b, c; first low), and anti-XCAP-H antibody (d, e, f; second low, short exposure: g, h; third low, long exposure). Bar, 10 μm. () Sperm chromatin was assembled in the mitotic extract (a, c, e) or interphase extract supplemented with 3.6 μM OA (b, d, f). After assembly, the reaction mixtures were supplemented with the indicated extra concentration of KCl at 22°C for 20 min, fixed, and stained with Hoechst. Bar, 10 μm