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doi: 10.1242/10.1242/jcs.00095

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Transcription factors RUNX1/AML1 and RUNX2/Cbfa1 dynamically associate with stationary subnuclear domains

¹ Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0106, USA
² Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0106, USA

View larger version (35K): [in a new window]   Fig. 1. Structure and expression of functionally active EGFP-RUNX fusion proteins. (A) A schematic of EGFP-RUNX expression constructs. EGFP proteins were fused to the N-termini of RUNX1, RUNX2 and RUNX2361. These constructs were generated using the restriction sites listed above each diagram as described in the Materials and Methods. EGFP-RUNX2361 lacks the C-terminal 152 amino acids of RUNX2 including the NMTS, but retains the RHD and NLS. Conserved functional domains of the fusion proteins are labeled as follows: EGFP: enhanced green fluorescent protein; QA: glutamine-alanine amino-acid stretch, specific to RUNX2; RHD: Runt homology domain; NLS: nuclear localization signal; NMTS: nuclear matrix targeting signal; VWRPY: conserved interacting sequence for TLE/Groucho, a repression protein. (B,C) Western blot analyses are shown of HeLa cell extracts after transfection with either EGFP-RUNX1, EGFP-RUNX2, EGFP-RUNX2361 or EGFP constructs. pcDNA3 empty vector, CMV-RUNX1 and CMV-RUNX2 expression vectors were used as controls. Proteins were detected using either a monoclonal EGFP antibody (B, top), a polyclonal RUNX1 antibody (C, right) or a polyclonal RUNX2 antibody (C, left). Cdk-2 was used as a control for equal protein loading (B and C, bottom panels). Positions of molecular weight markers are indicated on the right side of each blot. (D) CAT activity was assessed from HeLa cell extracts co-transfected with each reporter construct (OC promoter-CAT reporter and -83-OC-LUC) and each expression vector (EGFP, EGFP-RUNX2, EGFP-RUNX2361, RUNX2, EGFP-RUNX1 or RUNX1) as indicated. CAT values were normalized to the luciferase values and fold induction was calculated relative to the empty vectors. The results are means of 15 to 21 samples±s.e.m. P<0.0001.

View larger version (80K): [in a new window]   Fig. 5. RUNX1 and RUNX2 subnuclear foci are stationary in living cells within the nuclear space. SaOS-2 cells were transiently transfected with (A) EGFP; (B) EGFP-RUNX1; (C) EGFP-RUNX2 and (D) EGFP-RUNX2361 expression vectors. Time-lapse images were pictures captured at 0, 5, 10, 15 and 20 minutes (A) or 0, 5, 10, 20, and 30 minutes (B-D) using 100 milliseconds (A, B) or 200 milliseconds (C,D) exposure times. Arrowheads in B and C illustrate examples of stationary RUNX subnuclear domains. Scale bars equal 10 µm.

View larger version (73K): [in a new window]   Fig. 6. RUNX1 and RUNX2 dynamically associate with subnuclear foci in living cells in a C-terminus-dependent mechanism. SaOS-2 cells were transiently transfected with (A) EGFP-RUNX1; (B) EGFP-RUNX2; (C) EGFP-RUNX2361 and (D) EGFP expression vectors. Pre-bleached images are shown. The circles represent the entire photobleached areas in which the recovery rates were calculated. The black boxes in A and B represent the area encompassing the foci within the photobleached area in which the recovery rates were determined. The images shown were captured before photobleaching and 1, 3, 5, 10 and 45 seconds after photobleaching. The white bars correspond to a scale of 10 microns. (E) Recovery curves of the proteins are shown as relative fluorescence intensity vs. time. From these curves the half-time of recovery and percent immobile fraction were calculated as described in the Materials and Methods. The line at a relative intensity of 1.0 represents the fluorescence intensity before bleaching.

View larger version (55K): [in a new window]   Fig. 2. Absence of subnuclear organization of a mutant RUNX protein in fixed cells. SaOS-2 cells were transfected with either the wild-type EGFP-RUNX1, EGFP-RUNX2 or the mutant EGFP-RUNX2361 expression vectors. Both whole cell (WC) and nuclear-matrix—intermediate-filament (NMIF) preparations were performed as described in the Materials and Methods and show punctate foci for RUNX1 and RUNX2. The green fluorescence of EGFP was captured with a FITC filter (center images). Inserts show DAPI-stained nuclei (top left corners) and transmitted light photographs (lower right corners) of each cell. The scale bar equals 10 µm.

View larger version (38K): [in a new window]   Fig. 3. RUNX proteins localize to sites of active transcription. SaOS-2 cells were transiently transfected with (A) EGFP-RUNX1 and (B) EGFP-RUNX2. Nascent transcripts were labeled with BrUTP for 30 minutes. Confocal microscopy was used to capture images of the intrinsic green fluorescence of EGFP and BrUTP labeling using a rat -BrdU antibody (red). Merged images show, in NMIF preparations, colocalization of EGFP-RUNX1 or EGFP-RUNX2 with BrUTP incorporation (yellow) in a significant subset of foci. The images shown are 3D projections (top) and a center section (bottom). The scale bars equal 10 µm. Line scans below the images show the extent of colocalization across the center of the nucleus indicated by the white lines of the merged 3D projections in panels A and B.

View larger version (84K): [in a new window]   Fig. 4. RUNX1 and RUNX2 colocalize in common subnuclear domains. SaOS-2 cells were co-transfected with (A) EGFP-RUNX2 and Xpress (XPR)-RUNX2 (control) and (B) EGFP-RUNX1 and XPR-RUNX2. Whole cell (WC) and nuclear matrix-intermediate filament (NMIF) preparations were performed. The yellow fluorescence in the merged images indicates colocalization between the EGFP- and XPR-tagged RUNX proteins. Cells were stained with 0.05 µg/ml DAPI. Chromatin-extracted NMIF preparations do not present any DAPI staining as expected. Scale bars equal 10 µm.

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