The NMTS of AML-1B trans-activates heterologous reporter gene expression. The reporter gene is chromosomally integrated and is composed of multimerized GAL4-sites fused to the Adenovirus minimal E1A promoter that drives expression of luciferase activity. Data are expressed as fold-induction in response to CMV driven coexpression of fusion-proteins between the GAL4 DNA binding domain (aa 1–147; GAL) and C-terminal segments of AML-1B (GAL/AML 432–480 and GAL/AML 351–381), relative to cells transfected with the CMV-vector alone (control). Each bar represents the data from 5 experiments (2–3 replicates). The asterisks indicates statistically significant differences relative to the control (*P ≤ 0.0005; **P ≤ 0.005).

AML-1B/RNA polymerase II₀ sites are coupled with RNA synthesis. The HA/AML-1B construct was transfected into SAOS and ROS 17/2.8 cells. BrUTP labeling was performed with Triton X-100 permeabilized cells. Cells were double-stained with the B3 anti RNA polymerase II antibody and the BU33 anti-BrdUrd antibody. BrUTP (green) and RNA polymerase II₀ (red) single-color channels and the merged image are each shown, with yellow indicating colocalization of BrUTP and RNA polymerase II₀. The same extent of colocalization was observed in mock-transfected and untransfected cells (data not shown).

AML-1B is targeted to transcriptionally active nuclear matrix domains. (A and B) A subset of AML-1B and RNA polymerase II are colocalized in the nuclear matrix. Human osteosarcoma SAOS-2 cells were transiently transfected with a construct expressing HA-tagged CBFα2/AML-1B. In situ nuclear matrix samples were prepared 24 hr after transfection as described in Materials and Methods. HA/AML-1B was detected with an antibody against HA (green) (Left) and RNA polymerase II₀ with the B3 anti-RNA polymerase II antibody (red) (Center). The merged image is shown in the Right panel; yellow indicates colocalization of AML-1B with RNA polymerase II₀. The colocalization of AML-1B with RNA polymerase II₀ was evaluated by digital immunofluorescence microscopy (A) and by confocal microscopy (B) with a series of optical sections (0.5 μm intervals) through a single cell. B shows single-color and merged images (green, HA/AML-1B; red, RNA polymerase II; yellow, colocalization) for one of the optical sections. (C) Promoter recognition is required for the colocalization of AML-1B with RNA polymerase II. SAOS-2 cells were transfected with the HA-tagged L148D mutant that contains a point mutation in the runt homology domain and lacks DNA-binding activity. In situ nuclear matrix preparations were visualized as described in A (green, HA/AML-1B; red, RNA polymerase II; yellow, colocalization). The images shown were obtained by digital fluorescence microscopy. Similar images were obtained by directly photographing immunofluorescence signals (data not shown).

AML-1B does not colocalize with SC-35 RNA processing domain. SAOS cells were transfected with HA/AML-1B and evaluated by immunofluorescence analysis of the nuclear matrix in situ by using the HA antibody (green, AML-1B) and the SC-35 antibody (red, SC-35) that recognizes SC-35 RNA processing domain. In some cells, proximity of AML-1B and SC-35 domains is reflected by limited yellow staining.