Knock-down of methyl CpG-binding protein 2 (MeCP2) causes alterations in cell proliferation and nuclear lamins expression in mammalian cells

Abstract

Background: MeCP2 (CpG-binding protein 2) is a nuclear multifunctional protein involved in several cellular processes, like large-scale chromatin reorganization and architecture, and transcriptional regulation. In recent years, a non-neuronal role for MeCP2 has emerged in cell growth and proliferation. Mutations in the MeCP2 gene have been reported to determine growth disadvantages in cultured lymphocyte cells, and its functional ablation suppresses cell growth in glial cells and proliferation in mesenchymal stem cells and prostate cancer cells. MeCP2 interacts with lamin B receptor (LBR) and with Heterochromatin Protein 1 (HP1) at the nuclear envelope (NE), suggesting that it could be part of complexes involved in attracting heterochromatin at the nuclear periphery and in mediating gene silencing. The nuclear lamins, major components of the lamina, have a role in maintaining NE integrity, in orchestrating mitosis, in DNA replication and transcription, in regulation of mitosis and apoptosis and in providing anchoring sites for chromatin domains.In this work, we inferred that MeCP2 might have a role in nuclear envelope stability, thereby affecting the proliferation pattern of highly proliferating systems.

Results: By performing knock-down (KD) of MeCP2 in normal murine (NIH-3 T3) and in human prostate transformed cells (PC-3 and LNCaP), we observed a strong proliferation decrease and a defect in the cell cycle progression, with accumulation of cells in S/G2M, without triggering a strong apoptotic and senescent phenotype. In these cells, KD of MeCP2 evidenced a considerable decrease of the levels of lamin A, lamin C, lamin B1 and LBR proteins. Moreover, by confocal analysis we confirmed the reduction of lamin A levels, but we also observed an alteration in the shape of the nuclear lamina and an irregular nuclear rim.

Conclusions: Our results that indicate reduced levels of NE components, are consistent with a hypothesis that the deficiency of MeCP2 might cause the lack of a key "bridge" function that links the peripheral heterochromatin to the NE, thereby causing an incorrect assembly of the NE itself, together with a decreased cell proliferation and viability.

Figure 1

MeCP2 ablation causes a defect in cell proliferation with a delay in cell-cycle progression. (A) PC-3, (B) LNCaP and (C) NIH-3 T3 cells were transfected with siRNA MeCP2 or non-targeting siRNA CTRL oligos; MeCP2 ablation was checked at 5 and 7 days after transfection and PC-3, LNCaP and NIH-3 T3 cells, and cell proliferation was analysed (data are mean ± SD bars calculated from three independent experiments). (D) A representative FACS analysis of PC-3 treated with control siRNA (siRNA CTRL: plots 1, 2 and 3) or with siRNA against MeCP2 (siRNA MeCP2: plots 4, 5 and 6) that shows a delay in cell-cycle progression. (E) After 7 days of MeCP2 silencing was observed an enrichment of cells in sub-G₀/G₁ phase (M1 bar), a representative FACS analysis is shown in E1. The synthesis of three independent FACS experiments after 7 days of MeCP2 silencing is represented in bar chart E2 (statistics were performed using Student’s test. *: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells, P < 0.005). (F) A representative bi-parametric BrdU/PI FACS analysis of PC-3 cells after 7 days of MeCP2 silencing is shown in F1; the synthesis of three independent bi-parametric BrdU/PI FACS analysis of PC-3 cells after 7 days of MeCP2 silencing is represented in bar chart F2 (statistics were performed using ANOVA. *: significant difference in cell cycle phases between siRNA CTRL and siRNA MeCP2 PC-3 cells, P < 0.05).

Figure 2

MeCP2 silencing determines PC-3 cells suffering without triggering a severe apoptotic and senescence effects. (A) Fixed PC-3 cells, transfected with siRNA CTRL or siRNA MeCP2 oligos were analyzed by confocal microscopy. Phase contrast representative pictures are shown. (B)Annexin V-FITC/PI FACS analysis was performed in PC-3 cells after 5 and 7 days of MeCP2 RNAi. This staining reveals the amount of early apoptosis (LR = A⁺ + PI^-), late apoptosis (UR = A⁺ + PI⁺), living cells (LL = A^- + PI^-) and necrotic cells (UL = A⁺ + PI⁺); x-axis Annexin V stain signal, y-axis PI stain signal. The percentage of apoptotic cells (UR + LR) between control and siMeCP2 samples of different experiments is shown in AnnexinV positive cells bar chart (statistic were performed using ANOVA. *: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells after 5 days of silencing, P < 0.01. **: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells after 7 days of silencing, P < 0.001). (C) PC-3 cells were transfected with siRNA CTRL and siRNA MeCP2 oligos. After 7 days of transfection protein extracts were prepared and western blot against Cleaved-PARP (Asp214 89 kDa) and GAPDH, as loading control, was performed. (D) SA-β-galactosidase assay was performed at 7 days after the first MeCP2 silencing. Blue cells in (D1) untreated, (D2) siCTRL and (D3) siMeCP2 cells are shown. Bar chart shown the percentage of senescent PC-3 cells after 7 days of siRNA CTRL (0.23%) and siRNA MeCP2 (4.4%) calculated by the ratio between the number of senescent and non-senescent cells in different microscope fields (data are means ± SD bars). (E) Identification of SAHFs (senescence-associated heterochromatic foci). Fixed 7day MeCP2 silenced PC-3 cells and control cells were treated with antibodies against HP1γ (green) and H3K9me3 (red) proteins and stained with DAPI. Cells were analysed using fluorescence microscope and an enlargement of each representative senescence stain (indicated by the white arrow) is showed (box a, box b and box c). In bar chart is indicated the percentage of senescent PC-3 cells after 7 days of siRNA CTRL (1%ca) and siRNA MeCP2 (4%ca) calculated by the ratio between the number of SAHFs and total cells in different microscope fields (data are means ± SD bars).

Figure 3

MeCP2 functional ablation determines alteration of NE components expression. (A) PC-3, LNCaP and NIH-3 T3 cells were transfected with siRNA CTRL and siRNA MeCP2 oligos. Protein extracts were prepared at 5 and 7 days after transfection. Western blot assays against MeCP2 (70 kDa), lamin A/C (69 kDa, 62 kDa respectively), lamin B1 (68 kDa), LBR (58 kDa) and GAPDH (34 kDa), as loading control, were performed. (B) Quantitative RT-PCR showed that 7 days of MeCP2 silencing in PC-3 cells causes alteration of mRNA levels of LMNA, lamin B1 and LBR genes. mRNAs levels were normalized with respect to GAPDH housekeeping gene and were expressed as arbitrary units (statistics were performed using Student’s test. *: significant difference between siRNA CTRL and siRNA MeCP2, P < 0.001; °, significant difference between siRNA CTRL and siRNA MeCP2, P < 0.05). (C) Intracellular localization of endogenous lamin A and MeCP2 proteins. Fixed 7day MeCP2 silenced PC-3 cells and control cells were treated with antibodies against MeCP2 (green) and lamin A (red) proteins and analysed by confocal microscopy. Low levels of MeCP2 protein do not alter lamin A distribution, however an irregular nuclear rim is observed (C3-C6; C3a-C6a fluorescent and phase contrast images respectively). (D) In the line diagram is shown the local intensity distribution (diagonal white lines through the images) of MeCP2 (green), lamin A (red) in box1 (siRNA CTRL cell) and in box2 (siRNA MeCP2) respectively. (E) In the line diagram is shown the local intensity distribution of MeCP2 (green) and lamin A (red) of cell in box3.