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A specific targeting signal directs Runx2/Cbfa1 to subnuclear domains and contributes to transactivation of the osteocalcin gene

S. Kaleem Zaidi, Amjad Javed, Je-Yong Choi^*, André J. van Wijnen, Janet L. Stein, Jane B. Lian and Gary S. Stein

Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA 01655-0106, USA
^* Present address: Department of Biochemistry, School of Medicine, Kyungpook National University, 101 Dong-In Jung-Gu, Daegu 700-422, Korea

View larger version (64K): [in a new window]   Fig. 1. Endogenous Runx2 is associated with the nuclear matrix in osteoblastic cells. ROS 17/2.8 cells grown on gelatin-coated coverslips were processed for whole cell (WC) or nuclear matrix-intermediate filament (NM-IF) preparations and in situ immunofluorescence as described in Materials and Methods. Rabbit polyclonal antibody against Runx2 was used at a dilution of 1:200 (Javed et al., 2001). Secondary antibody used was Alexa 468 (goat against mouse) at a dilution of 1:800. The middle panel shows 4',6-diamidino-2-phenylindole (DAPI) staining, which is absent in NM-IF preparation as chromatin has been removed. Right panel shows phase contrast image of the same cell.

View larger version (36K): [in a new window]   Fig. 2. Runx2 resides in distinct subnuclear domains. HeLa cells transiently expressing Runx2 were processed for NM-IF preparation and double-label immunofluorescence to assess to what extent it colocalizes with nucleolin (A), coilin (B), SC35 (C) and PML bodies (D). Arrowheads point to distinct coilin and Runx2 domains (B) and PML bodies and Runx2 (D). Bars (2 µm) represent the relative magnification of the images.

View larger version (44K): [in a new window]   Fig. 3. Deletion mutants of Runx2 express at comparable levels and exhibit unaltered DNA binding activity. (A) Runx2 deletion mutants. The black box shows highly conserved runt homology domain (RHD). The dark gray box represents the amino acid sequences in Runx2 homologous to the NMTS of Runx1 whereas the C-terminal light gray box represents the highly conserved VWRPY domain. (B) HeLa cells grown in 100 mm plates were transfected at 50-60% confluence with 10 µg of the expression construct for each of the mentioned deletion mutants. The cells were harvested 24 hours after transfection in direct lysis buffer and proteins were separated by SDS-PAGE. The western blotting was carried out as described. The blots were incubated with monoclonal antibody against HA tag (Santa Cruz; dilution 1:3000) followed by incubation with HRP-conjugated secondary antibody raised against mouse (Santa Cruz; dilution 1:2000) to detect the proteins. Cdk2 antibody (1:5000) was used as an internal control. (C) HeLa cells transiently expressing mentioned deletion mutants were processed for nuclear extract preparation. Radioactively labeled Runx consensus sequence was used as probe. The mentioned proteins were used in increasing concentrations, that is, 2, 4 and 8 µg in electrophoretic mobility shift assay; EV (empty vector) indicates that cells were transfected with vector backbone.

View larger version (105K): [in a new window]   Fig. 4. The region responsible for subnuclear targeting of Runx2 resides in the C-terminus. ROS 17/2.8 cells grown on gelatin-coated coverslips were transfected with (A) Runx2 (1-528) (B) Runx2 (1-516) and (C) Runx2 (1-376). The cells were then processed for NM-IF preparation and in situ immunofluorescence. The coverslips were incubated with monoclonal antibody against HA tag (1:3000) followed by incubation with Alexa 568 conjugated secondary antibody (1:800) to detect the expressed proteins. Images were taken using Zeiss Axioplan digital microscope and Metamorph software was used for bio imaging. Bars (2 µm) represent the relative magnification of the images.

View larger version (95K): [in a new window]   Fig. 5. Runx2 has a functional NMTS. ROS 17/2.8 cells grown on gelatin-coated coverslips were transfected with (A) Runx2 (400-467) (B) Runx2 (397-434) and (C) Runx2 (433-467). The cells were then processed for NM-IF preparation and in situ immunofluorescence as for Fig. 4. Bars (2 µm) represent the relative magnification of images.

View larger version (38K): [in a new window]   Fig. 6. Subcellular fractionation of HeLa cells expressing deletion mutants of Runx2. HeLa cells expressing mentioned deletion mutants were subjected to biochemical fractionation as detailed in Materials and Methods. The fractions were processed for SDS-PAGE followed by western blotting. Monoclonal antibody against HA tag was used to detect the expressed proteins.

View larger version (48K): [in a new window]   Fig. 7. Conservation of the nuclear matrix-targeting signal in vertebrate species. Amino acid sequence alignments of the indicated Runx2 (Cbfa1/AML3), Runx1 (Cbfa2/AML1), and Runx 3 (Cbfa3/AML2/PEBP2A) proteins from different vertebrate species including two species of fish (Danio rerio (Danio), zebrafish; Oryzias latipes (Oryzi), Japanese killifish or medaka). Alignments were performed using CLUSTALW and graphically depicted using the GeneDoc software developed by K. B. Nicholas and H. B. Nicholas. Amino acids were colored on the basis of similarities in physiochemical properties (H,K,R, dark blue; N,Q, light blue; S,T, orange; L,I,V, dark gray, A,G, light gray; F,Y,W, maroon; M,C, yellow; P, white). None of the amino acid sequences contains negatively charged amino acids (D,E).

View larger version (38K): [in a new window]   Fig. 8. (A) The NMTS of Runx2 is functionally autonomous. HeLa cells grown on coverslips were transfected with Gal4 or Gal4-NMTS Runx2 and the cells were then processed for NM-IF preparation and in situ immunofluorescence as described. The expression of the proteins was detected by monoclonal antibody against Gal4 DBD (1:1000). (B) The NMTS of Runx2 can target a heterologous protein to the NM-associated Runx2 foci. ROS 17/2.8 cells grown on gelatin-coated coverslips were transfected with Gal4-NMTS Runx2 and were processed for double immuno labeling for endogenous Runx2 and over-expressed Gal4 DBD constructs. (C) The NMTS of Runx2 exhibits mild transactivation. The HeLa cells stably transfected with luciferase reporter carrying five Gal4 binding sites upstream of transcription initiation site were transiently transfected with Gal4 DBD or Gal4-NMTS. Cells were harvested in reporter lysis buffer 24 hours after transfection and assayed for luciferase activity as described.

View larger version (23K): [in a new window]   Fig. 9. Transactivation of bone-tissue-specific marker, osteocalcin, is dependent on association of Runx2 with nuclear matrix. HeLa cells grown on six- well plates were transfected with 0.1 µg of the mentioned expression constructs along with 1 µg p208 OC CAT and 0.05 µg RSV-Luc as internal control for transfection efficiency. Cells were harvested in 300 µl of 1x Reporter Lysis Buffer (Promega) and the lysates were processed for CAT and luciferase assays as described. The CAT values were normalized with respective luciferase values and were plotted as fold activity relative to empty vector control. The graph is representative of three independent experiments each with n=6 (±s.e.m.). An example of a typical CAT assay is shown.

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