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First published online 22 April 2008
doi: 10.1242/jcs.023119

Journal of Cell Science 121, 1613-1623 (2008)
Published by The Company of Biologists 2008
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Zfp64 participates in Notch signaling and regulates differentiation in mesenchymal cells

Kei Sakamoto*, Yoshihiro Tamamura, Ken-ichi Katsube and Akira Yamaguchi

Section of Oral Pathology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan


Figure 1
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  		Fig. 1. Illustration of the protein-domain organization of Zfp64. The numbers denote zinc-finger motifs. Zinc-finger motifs 6-9 of isoform-D are highly homologous to the zinc-finger motifs 3-6 of the other isoforms, and are labeled 3', 4', 5', 6' (using the respective numbers). Zinc-finger motifs 5 and 10-13 of isoform-D are labeled 2', 7', 8', 9', 10'. The yeast-two-hybrid screening yielded the isoform-D-specific C-terminal fragment (Zfp-C'). A construct that consists of the region common to all the splicing variants (Zfp-N) and a construct that consists of the C-terminal-fragment specific for isoforms A, B and C (Zfp-C) were created and used in the yeast-two-hybrid assay.

 

Figure 2
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  		Fig. 2. Zfp64 associates with NICD. (A) Yeast-two-hybrid assay. The PEST-containing region of Notch1 interacts with Zfp64 and its C-terminal region (Zfp-C'), and also with the C-terminal region of the other Zfp64 isoforms (Zfp-C); this was observed by formation of blue colony. Zfp-N, which is the N-terminal region of all the Zfp64 isoforms, showed no interaction with the PEST-containing region. The mock vector and lamin C (Lam) showed no interaction with Zfp64. (B) Translation of the genes fused with a GAL4 activation domain in each yeast transformants was confirmed by Western blot analysis using anti-HA antibody. Translation of the genes fused with a GAL4 DNA-binding domain was confirmed using anti-Myc antibody. (C) Zfp64 associates with the PEST-containing region of Notch1. HA-tagged Zfp64 and Myc-tagged PEST were co-synthesized in vitro using a rabbit reticulocyte lysate system. Immunoprecipitation of the PEST-containing region using anti-Myc antibody revealed co-precipitation of Zfp64. (D) Zfp64 associates with NICD in cells. HA-tagged Zfp64 and N-terminal FLAG-tagged NICD or NICD-p were co-transfected into HEK293 cells. Protein extraction and immunoprecipitation were performed in three different lysis buffers: RIPA, RIPA with EDTA, and RIPA with zinc acetate. Immunoprecipitation of NICD using anti-Flag antibody revealed the association of Zfp64 and NICD in RIPA with zinc acetate. A small amount of Zfp64 co-precipitant was detected in RIPA and the least amount of co-precipitant was detected in RIPA with EDTA. Zfp64 also co-precipitated with NICD-p in RIPA with zinc acetate. (E) Endogenous Zfp64 co-precipitated with adenovirally delivered NICD. U2OS cells were transfected with AdFlg-NICD-V5 and immunoprecipitation with anti-FLAG antibody was performed.

 

Figure 3
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  		Fig. 3. Zfp64 is expressed in a broad range of mesenchymal tissues. (A,B) Whole-mount in-situ hybridization using a (A) mouse dpc 11 embryo and (B) embryonic day 3.5 (HH stage 22) chick embryo. (C) Chick Hey1 expression. (D) Section view of B. (E) Zfp64 expression in an E6 (HH stage 28-29) embryo. (F) Section view of E. (G,H,I) In-situ hybridization to embryonic day 18.5 mouse embryo. Zfp64 is expressed in (G) the basal layer of the dermis and subepithelial mesenchymal cells, (H) chondrocytes and (I) osteoblasts. (J,K,L) Immunohistochemical staining of an embryonic day 18.5 mouse embryo using anti-Zfp64 antibody shows nuclear staining in an expression pattern similar to that seen in G-I. (M) Zfp64 expression in adult organs. Ubiquitous expression of Zfp64 was observed by RT-PCR. (N) Zfp64 expression in mesenchymal cell lines, measured by real-time PCR. *, Since U2OS cells are a human cell line, the PCR primers were different from those used for the other cell lines. (O) Western blot of C2C12 or U2OS cell lysates using anti-Zfp64 antibody. At least two bands of different molecular mass were observed. The band of the higher molecular mass appears to correspond to isoform A and/or C, and the small protein appears to correspond to isoform B and/or D of Zfp64. (P) Immunocytostaining of C2C12 and U2OS cells using anti-Zfp64 antibody.

 

Figure 4
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  		Fig. 4. Zfp64 upregulates Hes1 and Hey1 expression. (A,B) Dose-dependent activation of the Hes1 and Hey1 promoters by Zfp64. Graded amounts of Zfp64 plasmid were co-transfected with Hes1-luc or Hey1-luc reporter plasmids. Data represent the fold-inductions relative to that with mock vector transfection. All the error bars denote standard errors. (C,D) The activation of the Hes1 or Hey1 promoter by Zfp64 was similar to the level obtained with constitutively active Notch constructs. (E,F) NICD and Zfp64 additively activate the Hes1 and Hey1 promoters. Graded amounts of NICD and Zfp64 were co-transfected with the reporter plasmid and the luciferase activity was measured. (G,H) Additive effects of Zfp64 on NICD-p were weak (compare with Fig. 4E,F). (I,J) Zfp64 promoted the expression of Hes1 and Hey1. U2OS cells were transfected with AdGFP or AdZfp64. Total RNA was extracted 24 hours after infection and real-time RT-PCR analysis was performed. Expression levels of Hes1 and Hey1 were normalized to those of 18S rRNA. (K,L) Cells expressing Zfp64 reacted more robustly to Notch signal stimulation with Delta1. U2OS cells were transfected with AdGFP (U2) or AdZfp64 (U2(Z)), co-cultured with one-tenth of the number of HeLa cells (H) or HeLa cells stably transfected with Delta1 (H(Dl)). Real-time PCR analysis was performed after 2 days. Three independently conducted experiments gave similar results and representative data are shown. (M,N) Zfp64 is recruited to the Hes1 and Hey1 promoters. HA-tagged Zfp64 was transfected into U2OS cells with or without NICD, and the samples were subjected to a ChIP assay using primers for the Hes1 or Hey1 promoter sequence.

 

Figure 5
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  		Fig. 5. Zfp64 promoters. (A) The sequence of the putative promoter of human Zfp64 and the consensus transcription-factor-binding motifs. White and black squares indicate the core elements in the forward and reverse orientations, respectively. (B) Interspecies comparison of Zfp64 promoters. The transcription-factor-binding motifs are indicated by white squares (forward orientation) or black squares (reverse orientation).

 

Figure 6
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  		Fig. 6. Runx2 upregulates Zfp64 expression. (A) Luciferase activity assay. The Zfp64 promoter was activated by Runx2 in a dose-dependent manner. (B) MC3T3-E1, C2C12, and RD-C2 Runx2-deficient cell lines were transfected with AdGFP or AdRunx2. Zfp64 expression was measured by real-time RT-PCR, and shown as Runx2/GFP ratio (means ± s.e.). *P<0.05. (C) The distal ose2 element of the human Zfp64 promoter is crucial for its transactivation by Runx2. Deletions or mutations were introduced into the Zfp-luc reporter construct as illustrated, which was co-transfected with the mock or Runx2 expression plasmid into U2OS cells for a luciferase activity assay. (D) MC3T3-E1 cells or RD-C2 cells were treated with 100 ng/ml of human recombinant BMP2 protein. Expression of Runx2, Zfp64, Hey1 and Hes1 was examined by real-time RT-PCR and was expressed as fold-increase (mean ± s.e.) versus controls without BMP2. *P<0.05. ND, not detected.

 

Figure 7
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  		Fig. 7. Zfp64 inhibits myogenesis and promotes the expression of osteogenic marker genes. (A) Zfp64 and NICD inhibit the expression of muscle-specific cytoskeletal proteins. Tet-on-NICD-C2C12 cells were treated as described in Results and western blot analysis was performed. The band specific for desmin is shown by the arrow and the nonspecific band is indicated by an asterisk. (B) siRNA-mediated gene knockdown efficiency. Left. C2C12 cells were co-transfected with AdZfp64 and AdsiZfp64 in the indicated ratios. Western blot analysis was performed 1 day after transfection. (Right) C2C12 cells were transfected with mock or AdsiZfp64. Real-time PCR was performed 3 days after transfection. (C) Zfp64 and NICD inhibit the expression of myogenic transcription factors. Tet-on-NICD-C2C12 cells were treated as described in Results and real-time RT-PCR was performed. Data shown are representative of two independently conducted experiments (mean ± s.e.). (D) Myotube formation four days after myogenic induction, visualized by immunofluorescent staining using antibody against sarcomeric actin. (E) Alkaline phosphatase (ALP) activity of C2C12 cells increased by Zfp64. C2C12 cells were transfected with AdGFP, AdZfp64 or AdsiZfp64. Twelve hours after transfection, cells were treated with 20 ng/ml human recombinant BMP2 protein for 3 days and ALP activity was evaluated. (Top) ALP staining of cells. (Bottom) Relative ALP activity measured using pNPP substrate. Data are expressed as the mean ± s.e. of triplicates. (F) C2C12 cells were treated as described in E, then the expression of type I collagen (Col1a1), osteocalcin (Ocn) and osteopontin (Opn) was examined by real-time RT-PCR. Multiple independently conducted experiments were performed and representative data are shown as the mean ± s.e. of triplicates. (G) Relative ALP activity of HDC cells transfected with AdGFP, AdZfp64 or AdHey1. Data are expressed as the mean ± s.e. of triplicates. (H) Relative expression level of Col1a1, Ocn and Opn of HDC cells transfected with AdGFP, AdZfp64 or AdsiZfp64 (mean ± s.e.).

 

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© The Company of Biologists Ltd 2008