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. 2018 Feb;32(2):1108-1119.
doi: 10.1096/fj.201700780R. Epub 2018 Jan 3.

The histone demethylase KDM5A is required for the repression of astrocytogenesis and regulated by the translational machinery in neural progenitor cells

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The histone demethylase KDM5A is required for the repression of astrocytogenesis and regulated by the translational machinery in neural progenitor cells

Sun-Young Kong et al. FASEB J. 2018 Feb.

Abstract

Histone demethylases are known to play important roles in the determination of the fate of stem cells and in cancer progression. In this study, we show that the lysine 4 of histone H3 (H3K4), lysine-specific demethylase 5A (KDM5A) is essential for the repression of astrocyte differentiation in neural progenitor cells (NPCs), and its expression is regulated by translational machinery. Knockdown of KDM5A in NPCs increased astrocytogenesis, and conversely, KDM5A overexpression reduced the transcriptional activity of the Gfap promoter. Induction of astrocytogenesis by ciliary neurotrophic factor (CNTF) or small interfering RNA-induced knockdown of KDM5A decreased KDM5A recruitment to the Gfap promoter and increased H3K4 methylation. The transcript level of Kdm5a was high, whereas KDM5A protein level was low in CNTF induced astrocytes. During astroglial differentiation, translational activity indicated by the phosphorylation of eukaryotic translation initiation factor (eIF)4E was decreased. Treatment of NPCs with the cercosporamide, a MAPK-interacting kinases inhibitor, reduced eIF4E phosphorylation and KDM5A protein expression, increased GFAP levels, and enhanced astrocytogenesis. These data suggest that KDM5A is a key regulator that maintains NPCs in an undifferentiated state by repressing astrocytogenesis and that its expression is translationally controlled during astrocyte differentiation. Thus, KDM5A is a promising target for the modulation of NPC fate.-Kong, S.-Y., Kim, W., Lee, H.-R., Kim, H.-J. The histone demethylase KDM5A is required for the repression of astrocytogenesis and regulated by the translational machinery in neural progenitor cells.

Keywords: H3K4 demethylase; MAPK-interacting kinase; NSC differentiation; eIF-4E; epigenetics.

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Conflict of interest statement

This research was supported by the Chung-Ang University Excellent Student Scholarship and the National Research Foundation of Korea Grant NRF2017R1A1A1A05000876 (to H.J.K.), funded by the South Korean government. The Alabama Neuroscience Blueprint Core was supported by the U.S. National Institutes of Health, National Institute of Neurological Disorders and Stroke Grants NS39055 and NS057098. The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
KDM5A suppresses astrocyte differentiation in rat fetal NPCs. A) The specificity of Kdm5a, Kdm5b, and Kdm5d siRNA in NPCs was validated by real-time PCR. NPCs were nucleofected with Kdm5a, Kdm5b, Kdm5d, or control siRNA, and cells were cultured in the presence of EGF and FGF2 for 2 d. Kdm5a, Kdm5b, and Kdm5d mRNA expression levels were normalized to that of Gapdh,, and compared between siRNA-treated and control NPCs. B) Gfap, Olig2, Tubb3, and Nes mRNA expression levels were determined by real-time PCR. NPCs were nucleofected with Kdm5a, Kdm5b, Kdm5d, or control siRNA, and then cultured for 2 d in the presence of growth factors. mRNA expression levels were normalized to that of Gapdh and compared between siRNA-treated and control NPCs. Results are expressed as means ± sem of results of 3 independent experiments. *P < 0.05, **P < 0.01 (Student’s t test). C) Luciferase reporter constructs. The GFAP luciferase reporter construct comprises a human Gfap promoter fragment (−2163 to +47) inserted into the pGL3-Basic vector (empty luciferase reporter construct). D) Relative luciferase activity was measured 2 d after cotransfection of HEK293T cells with pcDNA3-HA-KDM5A or empty-pcDNA3, with the firefly GFAP luciferase-reporter (GFAP-LUC) or empty luciferase-reporter (Empty-LUC) construct, and the Renilla luciferase reporter vector. Firefly luciferase activity was normalized to Renilla luciferase activity, and then to activity exhibited by cells that were cotransfected with pcDNA3 and Empty-LUC. Data represent means ± sem of the results of 3 independent experiments. *P < 0.05 (Student’s t test). KDM5A levels in HEK293T cells transfected with GFAP-LUC and either the pcDNA3-HA-KDM5A or pcDNA3 vector were determined by Western blot analysis (2 d after transfection) (top). GAPDH was used as a loading control. E) KDM5A, GFAP, GALC, TUBB3, and GAPDH levels in NPCs nucleofected with either Kdm5a or control siRNA, and cultured (3 d) in the presence of EGF and FGF2, were determined by Western blot analysis. GAPDH was used as the loading control. F) Immunocytochemical data showing GFAP (red), GFP (green), and DAPI nuclear staining (blue) in NPCs nucleofected with either Kdm5a or control siRNA with the pmaxGFP vector and cultured (3 d) in the presence of mitogens. Arrows: GFAP/GFP double-positive cells. Scale bar, 50 μm. G) Quantitative analysis of GFAP/GFP double-positive cells among the total GFP-positive cells. The number of GFAP/GFP-positive cells detected in Kdm5a siRNA-treated NPCs was divided by that of control siRNA-treated NPCs. Data represent means ± sem of results of 3 independent experiments. **P < 0.01 (Student’s t test).
Figure 2.
Figure 2.
KDM5A is recruited to the Gfap promoter during proliferation and reduces the H3K4me3 level. A) Primers designed for the Gfap promoter regions. P1 includes the predicted KDM5A binding motif (CCGCCC), P2 contains a STAT binding site, P3 comprises a randomly selected site around position −1000, and P4 contains the TSS. B) Gfap expression was evaluated by real-time RT-PCR. NPCs were cultured (24 h) in the presence of EGF/FGF2, or CNTF without EGF/FGF2. Gapdh was used as the internal control. Data represent means ± sem of results of 3 independent experiments. *P < 0.05 (Student’s t test). C, D) ChIP analysis of H3K4me3 (C) and KDM5A (D) levels in the Gfap promoter regions (P1–4) in NPCs cultured (12 h) with EGF/FGF2 or CNTF only. Enrichment relative to input is shown as means ± sem of results in 3 independent experiments. *P < 0.05, **P < 0.01 (Student’s t test). EG) ChIP analysis of KDM5A (E), H3K4me3 (F), and H3K9me2 (G) levels in the Gfap promoter regions (P1–4) in NPCs nucleofected with either Kdm5a or control siRNA, then cultured (2–3 d) in EGF/FGF2. Enrichment relative to input is shown as means ± sem (n = 3, E; n = 5, F, G). *P < 0.05, **P < 0.01 (Student’s t test).
Figure 3.
Figure 3.
The increased Kdm5a transcript and decreased KDM5A protein expression during astrocyte differentiation is not caused by protein degradation. A) Relative Kdm5a levels were measured by real-time RT-PCR in NPCs cultured with EGF/FGF2 or CNTF only for 24 h. Gapdh was used as the internal control, and mRNA levels of EGF/FGF2-treated cells were set to 1. All values represent means ± sem of results in 3 independent experiments. **P < 0.01 (Student’s t test). B) KDM5A and GFAP protein levels in NPCs cultured for 24 h with EGF/FGF2 or CNTF were determined by Western blot analysis. GAPDH was used as the loading control. C, D) Western blot analysis of KDM5A and NICD levels in NPCs cultured for 24 h in EGF/FGF2 or CNTF only, and treated with either 5 μM MG132 (C), or 25 nM BTZ (D) for 6 h before lysis. NICD was used as a positive control for proteasome inhibition, and GAPDH was used as the loading control.
Figure 4.
Figure 4.
The discrepancy between Kdm5a and KDM5A expression in NPCs does not result from miRNAs activity. A) Schematic diagram of predicted miRNA binding sites in the Kdm5a 3′UTR. B) After expansion (1 wk) in EGF and FGF2, NPCs were dissociated, plated, and cultured (24 h) with EGF/FGF2 or CNTF only, before miRNA expression levels were determined by real-time PCR. RNU6 was used as the loading control. Data represent means ± sem of results in 3 independent experiments. *P < 0.05, **P < 0.01 (Student’s t test). C) Western blot analysis of KDM5A expression in NPCs nucleofected with the MDH1-PGK-GFP 2.0 vector expressing miR-9, miR-124, miR-181a, miR-181c, or no insert (negative control, NC), and cultured for 3 d in EGF and FGF2. GAPDH served as the loading control.
Figure 5.
Figure 5.
KDM5A translation is reduced during astrocytogenesis. A) Western blot analysis of KDM5A, p-eIF4E (Ser209), total eIF4E, p-ERK1/2 (Thr202/Tyr204), total ERK1/2, p-STAT3 (Tyr705), total STAT3, and GAPDH in NPCs cultured with EGF/FGF2 or CNTF only for the indicated periods. B) p-eIF4E, total eIF4E, KDM5A, GFAP, and GAPDH levels in NPCs were detected by Western blot analysis 3 d after treatment with 5 μM Cerco or DMSO (vehicle), in the presence of growth factors. C) Eif4e, Kdm5a, Gfap, Olig2, Tubb3, and Nes mRNA levels were measured by real-time PCR in NPCs cultured for 2 d with 5 μM Cerco or DMSO, in the presence of EGF/FGF2. The expression level of each mRNA was normalized to that of Gapdh. mRNA expression levels in DMSO-treated cells was set as 1. Data represent means ± sem of results in 3 independent experiments. *P < 0.05, **P < 0.01 (Student’s t test). D) Representative photomicrographs of NPCs treated 4 d with 5 μM Cerco or DMSO, in the presence of EGF/FGF2. Cells were immunostained for GFAP (red) and DAPI stained nuclei (blue). Scale bar, 50 μm. E) Quantitative analysis of astrocytes. The number of GFAP+ cells was divided by that of DAPI+cells. All values represent means ± sem of results in 3 independent experiments. **P < 0.01 (Student’s t test). F) p-ERK1/2, total ERK1/2, p-eIF4E, total eIF4E, KDM5A, and GFAP levels were assessed by Western blot analysis. NPCs were treated for 2 d with 50 μM U0126 or DMSO, in the presence of mitogens. GAPDH was used as the loading control. G) Kdm5a, Gfap, Olig2, Tubb3, and Nes expression was assessed by real-time PCR in NPCs cultured (2 d) with 50 μM U0126 or DMSO, in the presence of growth factors. mRNA expression in DMSO-treated cells was set as 1. Data represent means ± sem of results in 3 independent experiments. *P < 0.05, **P < 0.01 (Student’s t test).
Figure 6.
Figure 6.
KDM5A function and its expression control in proliferating NPC and differentiating astrocyte. A) NPCs proliferate in the presence of EGF and FGF2. The growth factors activate MEK/ERK/MNK signaling pathway that facilitates the phosphorylation of eIF4E, resulting in translation of KDM5A in NPCs. High levels of KDM5A demethylate H3K4 at transcription start site of Gfap promoter and repress astrocytogenesis and maintain cells as NPCs. B) CNTF induces astrocyte differentiation via JAK/STAT3 signaling in NPCs and inhibition of eIF4E mediated translation. During CNTF-induced astrocytogenesis, although Kdm5a transcription is up-regulated, its translation is reduced. In the absence of EGF and FGF2, ERK and MNK are no longer activated and subsequently eIF4E phosphorylation is reduced. The KDM5A translation is reduced and consequently, H3K4 methylation retained on Gfap promoter promotes transcription of Gfap and results in astrocytogenesis from NPCs.

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