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Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells - PubMed

  • ️Sun Jan 01 2017

Epigenetic up-regulation of ribosome biogenesis and more aggressive phenotype triggered by the lack of the histone demethylase JHDM1B in mammary epithelial cells

Alice Galbiati et al. Oncotarget. 2017.

Abstract

The alterations of ribosome biogenesis and protein synthesis play a direct role in the development of tumors. The accessibility and transcription of ribosomal genes is controlled at several levels, with their epigenetic regulation being one of the most important. Here we explored the JmjC domain-containing histone demethylase 1B (JHDM1B) function in the epigenetic control of rDNA transcription. Since JHDM1B is a negative regulator of gene transcription, we focused on the effects induced by JHDM1B knock-down (KD). We studied the consequences of stable inducible JHDM1B silencing in cell lines derived from transformed and untransformed mammary epithelial cells. In these cellular models, prolonged JHDM1B downregulation triggered a surge of 45S pre-rRNA transcription and processing, associated with a re-modulation of the H3K36me2 levels at rDNA loci and with changes in DNA methylation of specific CpG sites in rDNA genes. We also found that after JHDM1B KD, cells showed a higher ribosome content: which were engaged in mRNA translation. JHDM1B KD and the consequent stimulation of ribosomes biogenesis conferred more aggressive features to the tested cellular models, which acquired a greater clonogenic, staminal and invasive potential. Taken together, these data indicate that the reduction of JHDM1B leads to a more aggressive cellular phenotype in mammary gland cells, by virtue of its negative regulatory activity on ribosome biogenesis.

Keywords: JHDM1B; cancer cells; histone modification; rDNA; ribosome biogenesis.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. JHDM1B knock-down is associated with histone mark modification

(A and B) JHDM1B mRNA expression measured by real-time RT-PCR after 3 and 6 days of TRC administration in MDA-MB-231 sh1-JHDM1B (A) and MCF10 sh1-JHDM1B (B). Data were analyzed by paired Student's T-test: *P < 0.05; **P < 0.01; ***P < 0.001, (error bars, SEM). (C) Western blot analysis of the H3K4me3, H3K36me2 and total H3 histone levels in purified histones from MDA-MB-231 sh1-JHDM1B and MCF 10A sh1-JHDM1B cells. (D) Densitometry of the gels shown in Figure 1C. The band densities corresponding to the H3K4me3 and H3K36me2 were normalized to those of the total H3 histone and subsequently data were normalized on the relative controls (-TRC). Results were analyzed by paired Student's T-test *P < 0.05, (N = 3, error bars, SEM).

Figure 2
Figure 2. JHDM1B knock-down causes a surge in 45S pre-RNA synthesis and processing

(A) Evaluation of the neo-produced and processed 45S pre-rRNA in MDA-MB-231 sh1-JHDM1B. Cells were grown in medium supplemented with 5-ethynyl uridine for 1 h (pulse), or additionally grown in the presence of an excess of non-modified uridine for 2 h (chase), in order to evaluate respectively the neo-synthesized and processed 45S pre-rRNA by real time RT-PCR. (B) Evaluation of the neo-produced and processed 45S pre-rRNA in MCF 10A sh1-JHDM1B. Cells were grown in medium supplemented with 5-ethynyl uridine for 2 h (pulse), or additionally grown in the presence of an excess of non-modified uridine for 2 h (chase), results were analyzed by paired Student's T-test; *P < 0.05; **P < 0.01; ***P < 0.001, (N = 6, error bars, SEM). (C) 5-fluoro uridine incorporation in nascent RNA in MDA-MB-231 sh1-JHDM1B (left) and average fluorescent nucleolar area (right). (D) 5-fluoro uridine incorporation in nascent RNA in MCF 10A sh1-JHDM1B (left) and average fluorescent nucleolar area (right). Dapi staining of nuclei (left blue signal), anti-mouse Alexa-488 (center green signal), merge of the two channels (right). Results were analyzed by unpaired Student's T-test *P < 0.05 (error bars, SD).

Figure 3
Figure 3. JHDM1B KD causes a modulation of the H3K36me2 mark at the rDNA level

(A) Chromatin immunoprecipitated with an anti-H3K36me2 in MDA-MB-231 sh1-JHDM1B control (white-filled) and KD cells (black-filled). (B) Chromatin immunoprecipitated with normal rabbit IgG in MDA-MB-231 sh1-JHDM1B control (white-filled) and KD cells (black-filled). (C) Chromatin immunoprecipitated with an anti-H3K36me2 in MCF10A sh1-JHDM1B control (white-filled) and KD cells (black-filled). (D) Chromatin immunoprecipitated with normal rabbit IgG in MCF10A sh1-JHDM1B control (white-filled) and KD cells (black-filled). Quantification obtained by real-time RT-PCR with specific primers for different regions within ribosomal genes: promoter, and three sequences located 4 (H4), 8 (H8), and 13 (H13) kb downstream of the start transcription site, one sequence located in the non transcribed spacer (H30) and one sequence located in the IGS region. Data were expressed as a % of the input without further normalization or normalized over the % of input of rDNA promoter in control cells. The statistical analysis was by paired Student's T-test *P < 0.05; **P < 0.01 (N = 3, error bars, SEM).

Figure 4
Figure 4. MassARRAY EpiTYPER analysis of KD (black-filled) and control cells (white-filled)

(A and B) JHDM1B knock-down changes the methylation status of ribosomal genes: (A) MDA-MB-231 sh1-JHDM1B; (B) MCF 10A sh1-JHDM1B. Statistical analysis was by paired Student's T-test *P < 0.05; **P < 0.01; ***P < 0.001, (N = 5, error bars, SEM).

Figure 5
Figure 5. Polysome profile analysis of MDA-MB-231 sh1-JHDM1B cells

(A) Polysome profiles of control (black line) and KD cells (red line) obtained by measuring the absorbance at 254 nm in a continuous 15%–50% sucrose gradient. Numbers correspond to the sedimentation coefficients as follows: pre-polysomial 1) < 40 S; 2) 40 S; 3) 60 S; 4) 80 S, and post-polysomial > 80 5) disomes; 6) trisomes; and 7) the remaining polysome fractions. (B) Denaturing formaldehyde 1% agarose gel loaded with the total RNA extracted from each fraction obtained from the polysomal profile (M: 10000 bp RNA marker). Polysome profiles and gel are representative images of 3 independent experiments.

Figure 6
Figure 6. JHDM1B silencing triggers a more aggressive phenotype in the tested cell lines

(A) Growth curve profiles of control MDA-MB-231 sh1-JHDM1B cells (dotted line) and KD cells treated with TRC (solid line). (B) Growth curve profiles of control MCF 10A sh1-JHDM1B cells (dotted line) and KD cells (solid line) (N = 3, error bars, SEM). (C and D) Colony numbers obtained from (C) MDA-MB-231 sh1-JHDM1B and (D) MCF 10 A sh1-JHDM1B (N = 3, error bars, SEM). (E and F) Number of mammospheres produced by MDA-MB-231 sh1-JHDM1B (E) and MCF 10A sh1-JHDM1B (F). (G and H) Matrigel invasion assay of MDA-MB-231 sh1-JHDM1B (G) and MCF 10A sh1-JHDM1B (H). All graphs in the figure were obtained averaging at least three independent experiments (and no internal normalization between different experiments was performed), p values were obtained by unpaired Student's T-test *P < 0.05; **P < 0.01; ***P < 0.001 (error bars, SEM). The images below provide a representative picture of the matrigel-coated filters after 16 h of invasion. Photographs of 5 different areas were acquired for each filter, at 10× magnification, and used for cell counts. (N = 6, error bars, SEM).

Figure 7
Figure 7. JHDM1B knock-down in MDA-MB-231 sh1-JHDM1B xenograft

(A) Representative images of AgNOR staining in MDA-MB-231 sh1-JHDM1B control tumor (upper panel) and TRC-treated mice (lower panel). Images show the presence of large nucleoli in JHDM1B KD xenograft. (B) Average nucleolar area measured in peripheral regions of 10 different tumors for each treatment. (C) Average percentage of large nucleoli (greater than 4 μm) in the total counted nucleoli. p values were obtained using the unpaired Student's T-test ***P < 0.001 (error bars, SD).

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