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. 2004 Oct;15(10):4356-68.
doi: 10.1091/mbc.e04-02-0138. Epub 2004 Jul 21.

Cell-specific nucleolar localization of TBP-related factor 2

Philippe Kieffer-Kwon  1 , Igor Martianov, Irwin Davidson
Affiliations

Cell-specific nucleolar localization of TBP-related factor 2

Philippe Kieffer-Kwon et al. Mol Biol Cell. 2004 Oct.

Abstract

TATA-binding protein (TBP)-related factor 2 (TRF2) is one of four closely related RNA polymerase II transcription factors. We compared the intracellular localizations of TBP and TRF2 during the cell cycle and mitosis in HeLa cells. We show that during interphase, endogenous or exogenously expressed TRF2 is located almost exclusively in the nucleolus in HeLa or Cos cells. TRF2 localization is not affected by stress or mitotic stimuli, but TRF2 is rapidly released from the nucleolus upon inhibition of pol I transcription or treatment by RNase. These results suggest that localization of HeLa TRF2 requires a nucleolar-associated RNA species. In contrast, in 3T3 fibroblast cells, exogenously expressed TRF2 localizes to the nucleoplasm. Constitutive expression of ectopic TRF2 in 3T3 cells leads to a prolonged S phase of the cell cycle and reduced proliferation. Together with previous data, our results highlight the cell-specific localization and functions of TRF2. Furthermore, we show that during cell division, HeLa TRF2 and TBP are localized in the mitotic cytoplasm and TRF2 relocalizes into the nascent nucleoli immediately after mitosis, whereas TBP reassociates with the chromatin. Although partially contradictory results have been reported, our data are consistent with a model where only small proportion of the cellular TBP remains associated with specific promoter loci during mitosis.

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Figures

Figure 4.
Figure 4.
Localization of TRF2 within the nucleolus. HeLa cells were double-labeled with the indicated antibodies (TRF2-UBF, or TRF2-fibrillarin, red and green, respectively). A representative focal plane is shown to illustrate the localization of each protein within the nucleolus. Magnification, 400×.
Figure 1.
Figure 1.
Distinct localizations of TBP and TRF2 in HeLa cells. (A) HeLa cells were labeled with antibodies against TBP or TRF2 as indicated. (B) HeLa cells were double-labeled with antibodies against TRF2 (red) or pol I (green) as indicated. (C) 20 μg of HeLa cell cytoplasmic (C), nuclear (N), or nucleolar-enriched fraction were separated by SDS-PAGE, and TBP and TRF2 were revealed by immunoblot using the 3G3 and 2A1 antibodies. (D) Cos1 cells labeled with antibodies against TBP or TRF2. (E) HeLa cells were double-labeled with antibodies against TFIIA (red) or TRF2 (green) as indicated. Representative nucleoli are indicated with arrows. Magnification, 40× in all panels.
Figure 2.
Figure 2.
Nucleolar localization of TLF in transfected cells. (A–D) The antibodies used for labeling are indicated above each lane. Left panel, the immunostaining; center panel, the corresponding Hoechst-stained nuclei; and right panel, the merged images. Representative nucleoli are indicated with arrows. Magnification, 40× in all panels.
Figure 3.
Figure 3.
(A–D) TRF2 is nucleolar during S and G2 phases of the cell cycle. Cells were synchronized with thymidine and aliquots of cells were analyzed every hour for 8 h after release. The FACS analysis at the indicated times after release is shown in the top half of each panel and the immunolabelling with anti-TRF2 antibody in the bottom half. In the 8-h panel a prophase cells is observed (arrow) where TRF2 is being released from the nucleolus.
Figure 5.
Figure 5.
TRF2 is not released from the nucleolus in response to stress signals. (A) HeLa cells were exposed to UV light as indicated and fixed 6 h after exposure. Representative apoptotic nuclei are indicated by the arrows. (B) A table summarizing TRF2 localization after the indicated treatments. The doses of each compound are indicated. Cells were fixed and labeled at the indicated times after treatments.
Figure 6.
Figure 6.
Release of TRF2 from the nucleolus after treatment with actinomycin D and RNase. (A) Cells were treated with actinomycin D for the indicated times and double-labeled with antibody against TRF2 (red) and fibrillarin (green). (B) Labeling of actinomycin D–treated cells with antibody against TBP. (C) Cells were treated with saponin or saponin and RNase as indicated and double-labeled with antibody against TRF2 and fibrillarin.
Figure 7.
Figure 7.
Localization of TRF2 in 3T3 cells. (A) Transfected 3T3 cells were labeled with antibody against TRF2 or Flag as indicated. Transfected cells expressing TRF2 could be observed with the anti-Flag antibody, whereas only background staining was seen with the anti-TRF2 antibody. (B) Immunoblots of extracts cell lines stably expressing Flag-TRF2. Ten micrograms of cell extract were used in each lane. Lane 1, extracts from transiently transfected cells; lane 2, un transfected cells; and lanes 3 and 4, extracts from stably cell lines. Flag-TRF2 was detected by either anti-TRF2 or anti-Flag as indicated. (C and D) Immunofluorescence of cell line 1 with anti-Flag or anti-TRF2 as indicated. Strong nuclear expression of TRF2 is observed using the Flag antibody, whereas only background staining was seen with the anti-TRF2 antibody.
Figure 8.
Figure 8.
Properties of TRF2-expressing 3T3 cells. (A) Growth curves of each cell line; 2.5 × 104 cells of each line were plated at day 0. Viable cells were counted 2, 4, 6, and 7 d after seeding. The results show the average of three different experiments. (B) FACS analysis of the cell cycle. The values represent the proportion of cells at each stage of the cycle. The results are averages of three experiments rounded to the nearest whole number. (C) Cells were exposed to UV irradiation (150 J · m-2) and the cell cycle analyzed by FACS after the indicated times. The values are averages of two experiments rounded to the nearest whole number.
Figure 9.
Figure 9.
Localization of TRF2, TBP, and TAF4 during mitosis. (A and B) Cells were labeled with antibody against TRF2 and counterstained with Hoechst. The stage of mitosis is indicated. (C and D) A similar analysis of mitotic stages with monoclonal antibodies against TBP and TAF4.
Figure 10.
Figure 10.
Exclusion of TBP and TAF4 from condensed mitotic chromatin. (A and B) Representative focal planes from confocal sections are shown. The left panel shows the antibody staining with the polyclonal rabbit antisera against TBP or mAb against TAF4 and the right panel the merge with the Hoeschst stained DNA. The mitotic stages are indicated.
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