Research ArticleCELL BIOLOGY

Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

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Science Advances  30 Oct 2020:
Vol. 6, no. 44, eabb3154
DOI: 10.1126/sciadv.abb3154
  • Fig. 1 CDK4/6 interacts with SMYD2 and regulates its phosphorylation.

    (A to C) Interactions of CDK4 (A) or CDK6 (B) with SMYD2, and SMYD2 with CDK4 or CDK6 (C) in RCTE cells were detected by immunoprecipitation (IP) and immunoblotting (IB). Immunoglobulin G (IgG) was used as a negative control. (D) Schematic of GST-SMYD2 fusion protein constructs (top), which was detected using Coomassie blue staining (middle). GST pull-down assays indicated the interaction of GST-SMYD2 fusion proteins with CDK4 and CDK6 (bottom). (E) GFP-tagged SET domain–deleted SMYD2 cannot pull down CDK4 and CDK6 in HEK293T cells. (F to I) The phosphorylation of SMYD2 was decreased in RCTE cells transfected with siRNAs to CDK4 (F), CDK6 (G), or both (H), and in RCTE cells treated with Abe (10 μM) (I). Knockdown of CDK4 decreased the expression of CDK6, and vice versa for CDK6 in these cells. The quantification analysis (n = 3) of band intensities was shown in the graphs (bottom), in that the density of each protein band was normalized to actin, and then the value was divided by the value of corresponding siRNA and vehicle band density to actin. The value of the control band to actin was set to 1.

  • Fig. 2 Targeting CDK4/6 decreased the methylation of histones mediated by SMYD2 in RCTE cells and SMYD2 also regulated the expression of CDK4 and CDK6.

    (A to D) Western blot analysis indicated that knockdown of CDK4 and CDK6 with siRNAs (A) or knockdown of SMYD2 with siRNA (B) as well as inhibition of CDK4/6 with Abe (C) or inhibition of SMYD2 with AZ505 (D) decreased the mono-, di-, and trimethylation of histone H3 at lysine 4 (H3K4) and lysine 36 (H3K36) in RCTE cells. The quantification and statistical analysis (n = 3) were shown in the graph (bottom). *P < 0.01 as compared to each control. (E and F) Knockdown (E) or inhibition (F) of SMYD2 decreased the mRNA and protein levels of CDK4 and CDK6 in RCTE cells, examined with qRT-PCR and Western blotting. *P < 0.01 as compared to each control (n = 3). ns, not significant. (G) SMYD2 bound to the promoter of CDK4 and CDK6. ChIP-qPCR analysis was performed with an SMYD2 antibody, or normal rabbit IgG in RCTE cells. (H) ChIP assay was performed with mono-, di-, and trimethylated H3K4 and H3K36 antibodies and normal rabbit IgG in RCTE cells.

  • Fig. 3 Depletion of Smyd2 promotes cilia assembly in mouse renal epithelial cells.

    (A) Immunofluorescence staining of primary cilia with α-acetyl-tubulin antibody (α-ac-tubulin) in primary renal epithelial cells isolated from kidneys of wild-type (WT) and Smyd2flox/flox:Ksp-Cre mice, in which Smyd2 was specifically knocked out (KO) by the kidney-specific promoter (Ksp)–driven Cre recombinase in renal epithelial cells and cultured in serum-free medium for 72 hours before subjected to staining. The percentage of ciliated cells and cilia length was measured and statistically analyzed. Scale bar, 5 μm. Error bars represent the SD. N values represent the numbers of cilia (left graph) and cells (right graph), respectively, for each group. (B to E) Knockdown of Smyd2 with siRNA (B) or inhibition of Smyd2 with AZ505 (C) as well as knockdown of CDK4/CDK6 with siRNAs (D) or inhibition of CDK4/CDK6 with Abe (E) in mIMCD3 cells resulted in longer cilia compared to control cells as examined by immunofluorescence with α-acetyl-tubulin and SMYD2 antibodies. Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers for each group.

  • Fig. 4 SMYD2 interacts with α- and γ-tubulin and methylates α-tubulin in vitro.

    (A) Coimmunoprecipitation of endogenous α- and γ-tubulin with SMYD2 in RCTE cells. IgG was used as a negative control. (B to D) Coimmunoprecipitation of endogenous α-tubulin (B), γ-tubulin (C), and β-tubulin (D) with overexpressed SMYD2 in HEK293T cells. GFP vector–transfected cells were used as a negative control. (E) GST pull-down assays were performed by incubation of GST-SMYD2 fusion protein with 1 μg of recombinant α- or γ-tubulin protein and immunoblotting with an α-tubulin (top) and γ-tubulin antibody (middle). The expression of GST-SMYD2 was detected with Coomassie blue staining (bottom). (F) The expression of GST-SMYD2 constructs was detected using Coomassie blue staining (top). GST pull-down assays of GST-SMYD2 fusion proteins incubated with 1 mg of cell lysate from RCTE cells and immunoblotting using α- and γ-tubulin antibodies (bottom). (G and H) In vitro methylation assay of α-tubulin (G), γ-tubulin (H), and recombinant SMYD2. Histone H3 was used as a positive control in the in vitro methylation assays. (I) Relative intensity of methylated α- and γ-tubulin bands (black box) compared to the intensity of methylated histone H3, which was set to 1 (open box), in the in vitro methylation assays.

  • Fig. 5 SMYD2 methylates α-tubulin at lysine-394 (K394) but not at K40.

    (A and B) Knockdown of SMYD2 with siRNA (A) or inhibition of SMYD2 with AZ505 (B) decreased methylation of α-tubulin at K394 but not at K40 as examined by Western blotting with our generated TubK40me3 and TubK394me3 antibodies in RCTE cells. The quantification and statistical analysis (n = 3) were shown in the graphs (bottom), in which the band density of control siRNA (A) and DMSO (B) was set to 1. (C) Western blot of the methylation of α-tubulin at K40 and at K394 with the TubK40me3 and TubK394me3 antibodies in RCTE cells transfected with Flag-tagged SMYD2 and GFP-tagged α-tubulin. The quantification and statistical analysis (n = 3) were shown in the graph (bottom). (D) Representative images of RCTE cells stained with TubK394me3 (left) or SMYD2 (right) and costained with α-acetyl-tubulin/γ-tubulin (red) antibodies and DAPI (blue). Scale bar, 5 μm. (E) Representative images of RCTE cells stained with TubK394me3 (left, green) or SMYD2 (right, green) and costained with α-acetyl-tubulin/γ-tubulin (red) and DAPI (blue). Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers in (D) and (E).

  • Fig. 6 Depletion of SMYD2 increases the expression and ciliary trafficking of IFT20.

    (A and B) Knockdown (A) and inhibition of SMYD2 (B) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. The quantification and statistical analysis (n = 3) were shown in the graph (bottom), as are also shown in (B), (C), (E), and (F). *P < 0.01 as compared to controls. (C) Overexpression of SMYD2 decreased the levels of IFT20 protein (left) and mRNA (right) as examined by Western blotting and qRT-PCR in HEK293T cells. (D) SMYD2 and H3K36me3 antibodies bound to the promoter of IFT20 in RCTE cells as examined with ChIP assay. (E and F) Knockdown (E) and inhibition of CDK4/6 (F) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. n = 3. (G) Representative images of RCTE cells stained with IFT20 and α-acetyl-tubulin/γ-tubulin antibodies and DAPI in the presence of AZ505 (middle), Abe (right), and vehicle (left). Scale bar, 5 μm. The statistical analysis was shown in the graph (right). Error bars represent the SD. N values represent cilia numbers.

  • Fig. 7 Primary cilia assembly was decreased in CDK4/6 and SMYD2 up-regulated breast cancer cells and cystic renal epithelial cells, whereas targeting CDK4/6 and SMYD2 restored cilia assembly in these cells.

    (A) Western blot of SMYD2, CDK4, CDK6, and IFT20 in normal mammary cells MCF10A and breast cancer cells, including MCF7, T47D, MDA-MB468, and MDA-MB231 cells. (B) Western blot of SMYD2, IFT20, CDK4, and CDK6 in PH2 and PN24 cells. (C) The quantification and statistical analysis (n = 3) were shown in the graphs corresponding to Fig. 6A (top) and Fig. 6B (bottom). (D to F) Representative images of T47D (top) and MDA-MB468 cells (bottom) (D) as well as PN24 cells (E and F) stained with SMYD2 (green) and α-acetyl-tubulin/γ-tubulin (red) and costained with DAPI (blue) in the presence of the SMYD2 inhibitor AZ505 (middle) and the CDK4/6 inhibitor Abe as well as vehicle. Scale bar, 5 μm. Statistical analysis of the percentage of ciliated breast cancer cells (n = 200) (P < 0.01) as well as the percentage of ciliated PN24 cells (n = 900) and their cilia lengths are shown in the graph (right). Error bars represent the SD.

  • Fig. 8 Targeting SMYD2 and CDK4/6 affects cilia-dependent Hedgehog signaling.

    (A and B) Representative images of RCTE (A) and PN24 (B) cells stained with GLI2 (green) and GLI3 (red), and costained with α-acetyl-tubulin/γ-tubulin (purple) antibodies and DAPI (blue). Scale bar, 5 μm. The statistical analysis of the cilia tip localization of GLI2 (left) and GLI3 (right) is shown in the graphs. *P < 0.01. Error bars represent the SD. n = 100 cilia for each group. (C and D) Western blot analysis of Ptch1 and GLI1 in RCTE cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (C) and the CDK4/CDK6 inhibitor Abe (D) in serum-free medium for 48 hours. Statistical analysis of the expression (n = 3) of Ptch1 and GLI1 proteins is shown in the graphs (right). (E and F) Western blot analysis of Ptch1 and Gli1 in PN24 cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (E) and the CDK4/CDK6 inhibitor Abe (F) in serum-free medium for 48 hours. Statistical analysis of the expression (n = 3) of Ptch1 and Gli1 proteins is shown in the graphs (bottom).

Supplementary Materials

  • Supplementary Materials

    Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

    Linda Xiaoyan Li, Julie Xia Zhou, Xiaodong Wang, Hongbing Zhang, Peter C. Harris, James P. Calvet, Xiaogang Li

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