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First published online December 31, 2003
doi: 10.1242/10.1242/jcs.00870

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The Yin Yang-1 (YY1) protein undergoes a DNA-replication-associated switch in localization from the cytoplasm to the nucleus at the onset of S phase

¹ Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4370, USA
² Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306-4300, USA

View larger version (18K): [in a new window]   Fig. 1. Incorporation of BrdU into synchronous populations of CHO cells at entry into S phase. Mitotic cells were pulse-labeled with BrdU for the last 30 minutes before the end of each interval. Fixed cells were subjected to fluorescence immunocytochemistry designed to visualize nuclear BrdU incorporation, indicative of S phase cells. FITC images across the top are from optical sections through individual cells, showing one example of a BrdU-negative cell (little to no fluorescence, 0 hours) and three examples of BrdU-positive cells with clear nuclear staining (3, 5 and 7.5 hours). The nucleus occupies most of the cell volume in these cells, and the cell periphery is not indicated. The percentage of intact cells (n=500 per time point) showing nuclear staining of BrdU is indicated at the bottom of the figure. The time after mitosis (in hours) is indicated at the top.

View larger version (94K): [in a new window]   Fig. 9. Loss of nuclear YY1 in response to inhibition of DNA synthesis, and recovery of nuclear YY1 with cell-cycle checkpoint inhibitors. Asynchronously growing CHO cells were fixed, immunostained and imaged with a confocal microscope. Images of immunodetectable YY1 (left column) and BrdU (middle column) are shown separately in gray scale and together as two-color overlay (red for YY1 and green for BrdU). Each row shows a field of cells from each of the four different treatments displayed at the same scale. (A-C) Untreated control cells. A subset of these cells are positive for both nuclear YY1 and BrdU staining (white arrows). (D-F) Aphidicolin-treated cells show loss of nuclear staining for both YY1 and BrdU. (G-I) Addition of the checkpoint inhibitor 2-aminopurine (2-AP) to aphidicolin-inhibited cells resulted in restoration of BrdU labeling and YY1 nuclear accumulation (white arrows). A few cells that did not overcome the block also failed to show YY1 (white arrowhead). (J-L) Addition of the checkpoint inhibitor caffeine (CAF) to aphidicolin-blocked cells had the same effect as 2-AP addition. Scale bars: 10 µm.

View larger version (37K): [in a new window]   Fig. 2. Increases in histone mRNA levels in vivo and YY1-histone alpha-element-binding activity in vitro upon entry into S phase. (A) Total RNA was isolated from stable transfectant CHO cells for each time interval indicated across the top (AS, asynchronous; numbers are hours after mitotic shake-off). A fragment of the mouse H3.2-614 gene (3'-end-labelled) was used as a radioactive probe in S1-nuclease protection assays, with 3 µg of total RNA per reaction. The positions of the probe fragments protected by the mouse and hamster histone transcripts (arrows) and the positions of markers (nucleotide number) are indicated. (B) An electrophoretic mobility-shift assay using the duplex histone alpha oligonucleotides as a probe was carried out with nuclear extracts from synchronous populations of CHO cells (lanes 0, 1, 3, 5 and 7.5; numbers are hours after shake-off) or from mouse myeloma cells (lane MM) as a positive control. The position of the complex (resulting from YY1 DNA-binding activity) and free probe are indicated.

View larger version (61K): [in a new window]   Fig. 3. YY1 mRNA and protein levels throughout the cell cycle. Numbers above the lanes indicate cell-harvest time points (hours after shake-off). (A) RNA gel blots of equal amounts of total RNA from synchronized CHO cells were incubated with radiolabeled cDNA clones for YY1, then reprobed with EF1- as a loading control. (B) Protein gel blots were incubated with anti-YY1 antibodies (YY1) or anti--tubulin (tubulin) and detected by ECL.

View larger version (56K): [in a new window]   Fig. 4. Accumulation of YY1 in two distinct subcellular locations, cytoplasm and nucleus. Optical sections of triple-label images are shown for two cells fixed at different time points after shake-off (3 hours, A-C; 7.5 hours, D-F). Cells were stained by indirect immunofluorescence and data collected by serial optical sectioning microscopy. For each image, a projection (1-1.2 µm thick in Z comprising 4-6 optical sections) from a middle region of the data stack is shown. DAPI images (A,D) reveal the nucleus (boundary indicated by long-dashed lines). YY1 was visualized in the rhodamine channel (B,E), and BrdU was visualized in the FITC channel (C,F). The nuclear periphery (long-dashed lines) and the cell periphery (short-dashed lines) are indicated, and the nuclear and cytoplasmic areas are indicated by `n' and `c', respectively.

View larger version (82K): [in a new window]   Fig. 5. Change in the localization of YY1 from cytoplasmic to nuclear upon entry into S phase. Synchronized CHO cells were fixed at the times indicated (top, hours after shake-off) and subjected to indirect immunofluorescence for visualization of the distribution of YY1 (E-H) and BrdU (I-L). 3D multiple-wavelength images were collected and are displayed as described in Fig. 4, and representative cells (one cell per column) are shown. Dashed lines indicate the cell periphery. YY1 signals appear mainly cytoplasmic at 1 hour (E), mainly nuclear at 3 (F) and 5 (G) hours, and then mainly cytoplasmic again at 7.5 hours (H). BrdU labeling of nuclei appears at 3 hours (J) and is strong throughout the nuclear volume at 5 (K) and 7.5 (L) hours. At the bottom (M-P), all three wavelengths are shown as a three-color (pseudo) merged images (blue for DAPI, green for Rhod/YY1, red for FITC/BrdU). Scale bars: 2 µm.

View larger version (16K): [in a new window]   Fig. 6. Cell cycle changes in YY1 distribution patterns. At least 100 cells per time point were chosen in the DAPI wave length, then classified by direct examination of YY1 staining as mainly cytoplasmic (solid diamond) or mainly nuclear (gray box).

View larger version (37K): [in a new window]   Fig. 7. Change in YY1 subcellular localization in HeLa cells. Synchronized HeLa S-3 cells were collected by mitotic shake-off at the time points indicated. Fixed cells were stained for YY1, imaged and presented as described in Fig. 4. Color-merge images show the nucleus (red, DAPI) and YY1 (green); dashed lines indicate the cell periphery. At late mitosis (A) and 1 hour after shake-off (B), YY1 appears mainly cytoplasmic. After 3 (C) and 5 (D) hours, most of the YY1 signals are located in the nucleus, where they appear as yellow-green in the merged images because of colocalization with red DAPI signal. Scale bars: 2 µm.

View larger version (40K): [in a new window]   Fig. 8. Cell-cycle changes in YY1 distribution observed by cofractionation assay. Cytoplasmic and nuclear extracts from synchronous populations of CHO cells were analyzed by western blot with anti-YY1 antibodies as described in Fig. 2B. Numbers above the lanes indicate the time of harvest (hours after shake-off). Cytoplasmic (Cyto.) or nuclear (Nucl.) extracts were loaded on an equal-protein basis (7 µg per lane).

View larger version (11K): [in a new window]   Fig. 10. Percentage of cells showing BrdU incorporation and nuclear localization of YY1 in response to aphidicolin and checkpoint inhibitors. The experiment was replicated three times, and for each treatment indicated at the bottom of the graph, at least 100 cells were scored. In the treatments with caffeine and 2-AP, all cells that showed BrdU incorporation also showed nuclear YY1.

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