Intranuclear targeting of AML/CBFalpha regulatory factors to nuclear matrix-associated transcriptional domains - PubMed (original) (raw)

. 1998 Feb 17;95(4):1585-9.

doi: 10.1073/pnas.95.4.1585.

S McNeil, S Pockwinse, J Nickerson, L Shopland, J B Lawrence, S Penman, S Hiebert, J B Lian, A J van Wijnen, J L Stein, G S Stein

Affiliations

Intranuclear targeting of AML/CBFalpha regulatory factors to nuclear matrix-associated transcriptional domains

C Zeng et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The AML/CBFalpha runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFalpha factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFalpha must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

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Figures

Figure 1

Figure 1

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).

Figure 2

Figure 2

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).

Figure 3

Figure 3

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).

Figure 4

Figure 4

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.

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