NPAT concentrates at a few foci in the nuclei of human cells. (A) Localization of NPAT in the indicated cell lines was examined by indirect immunofluorescence (IF) using an affinity-purified rabbit anti-NPAT antibody (R48). The antirabbit IgG secondary antibody was conjugated with Texas Red (Red). Nuclear DNA (nucleus) was stained with DAPI (blue). (B) Multiple NPAT antibodies recognize the same NPAT foci. WI38 cells were double stained with a mouse monoclonal NPAT antibody (DH3 or DH4) and an affinity-purified rabbit polyclonal NPAT antibody (R48 or R49). The staining from the mouse antibodies (green) and that from the rabbit antibodies (red) are completely overlapping (yellow). (C) Most WI38 and MRC5 cells have two or four NPAT foci (spots). WI38 and MRC5 cells were stained with R48, and the number of NPAT spots in each cell was counted. The mean results from three independent experiments and the standard deviations are shown. More than 200 cells were analyzed in each experiment.

There are four NPAT spots in S phase cells and two NPAT spots in non-S phase cells. (A) WI38 cells were incubated with 50 μM BrdU for 3.5 h, fixed, and stained with a mouse anti-BrdU antibody (red) and the rabbit anti-NPAT antibody R48 (green). DNA was stained with DAPI (blue). (B) WI38 cells were incubated with BrdU for 20 min and then fixed and stained as described in A. 100–150 cells were analyzed for the number of NPAT spots and BrdU staining status. The mean results from three independent experiments are depicted. The bars represent the standard deviations.

The localization of NPAT changes during the cell cycle. WI38 cells were arrested at G₀ by serum starvation as previously described (Tsai et al. 1993). At the time when the cells were released from the arrest, BrdU (final concentration 50 μM) was added into the culture medium. The cells were fixed, permeablized, and stained as described for Figure 2 at 6 h (G₁ phase), 19 h (S phase), 24 h (G₂), and 28 h (M phase) after release. The G₀ (serum-starved) cells were treated with BrdU for 24 h before being fixed. The BrdU staining for the cells in M phase is not shown.

NPAT overlaps with coiled bodies. U2OS cells were double stained with the rabbit anti-NPAT antibody (R48; red) and a mouse monoclonal antibody 5P10 (green; left panel) or with the mouse anti-NPAT monoclonal antibody (DH4; red) and a rabbit anti-PML antibody (green; right panel). The extent of overlap between NPAT and coilin varies from cell to cell and from cell line to cell line (data not shown).

NPAT associates with histone gene clusters on both chromosomes 1 and 6 in S phase. (A) The NPAT spots are associated with histone gene clusters on chromosome 1 and 6. WI38 cells were first stained with the rabbit anti-NPAT antibody (R48; red), fixed again, and hybridized with DIG labeled histone cluster–specific DNA probes (green). The nuclear DNA was stained with DAPI (blue). Top panels, images obtained with the probe specific for the histone cluster on chromosome 1; bottom panels, images with the probe specific for chromosome 6 cluster. For each probe, two cells, one with two NPAT spots (left panels) and one with four NPAT spots (right panels), are shown. Note that overlap of IF (red) and FISH (green) staining results in yellow dot. (B) All NPAT spots are associated with histone clusters in S phase. WI38 cells were first stained with the rabbit anti-NPAT antibody (R48; blue), fixed again, and hybridized with probes specific for chromosome 1 (green) and chromosome 6 (red) histone gene clusters. For easy viewing, the colors in the figure (except for the merged images) were shown as black and white. The results presented in this figure have been reproducibly observed in multiple independent experiments.

NPAT activates histone H4 transcription. (A) NPAT activates transcription from H4 promoters. U2OS cells were transfected with 1 μg pCMV (vector) or 1 μg pCMV–NPAT together with 50 ng pGLH4 (labeled as H4), pGLH4-1 (labeled as H4-1), pGL3 control (labeled as Control), pGL–b-bmyb (labeled as b-myb), or pGL–dhfr (labeled as dhfr) reporter construct. Fifty ng pCMV–lacZ was also co-transfected in each transfection. Thirty-six hours after transfection, cells were lysed and the activities of β-galactosidase and luciferase were assayed as described in Materials and Methods. The activities of the β-galactosidase were used to normalize the transfection efficiency among different samples. Fold of induction was calculated by comparing the luciferase activity from NPAT-transfected cells with that from the vector transfected cells. The figure shows the mean results and standard deviations from at least three independent experiments. (B) U2OS cells were transfected with 1 μg pCMV together with 50 ng pGLH4, pGLH4(80), pGLH4(65), pGLH4(40), pGLH4(D1), or pGLH4(M1) (as indicated as H4, 80, 65, 40, D1, and M1, respectively). Thirty-six hours after transfection, the cells were lysed and the β-galctosidase and luciferase activities were measured as described in A. The data represent the results from at least three independent experiments. (C) Activation of histone H4 transcription by NPAT is dependent on the H4 SSCS. U2OS cells were transfected with 1 μg vector or 1 μg pCMV–NPAT together with 50 ng of indicated reporter construct. Thirty-six hours after transfection, the samples were analyzed as described in A. H4, 80, 65,40, D1, and M1 represent the plasmids as described in B. The means and standard deviations from at least three independent experiments are depicted.

NPAT activates histone H4 transcription independent of its cell cycle effects. (A) U2OS or SAOS2 cells were transfected as described in Fig. 6A. The transfected U2OS and SAOS2 cells were lysed at 22 h and 36 h, respectively, after transfection. The samples were analyzed as described in Fig. 6A. The results from three independent experiments are shown. (B) U2OS cells, grown on 10-cm plates, were transfected with 6 μg pCMV (vector) or 6 μg pCMV–NPAT together with 300 ng pGLH4(65) and 300 ng pCMV–lacZ. Five hours after transfection, Nocadazole (final concentration 70 ng/mL) was added into the culture medium to arrest the cells in M phase. Eighteen hours later, cells in M phase were harvested by mitotic shake-off. The cells were washed twice with culture medium and replated. Two and one-half hours after replating, the distribution of the cells in the cell cycle was analyzed by FACS and the effect of NPAT on H4 transcription was analyzed as described in Fig. 6A. The mean results from two independent experiments are shown. (C) U2OS cells were transfected with 50 ng pGLH4 (65) together with pCMV vector (1 μg), p27 (100 ng), NPAT (0.5 μg), or NPAT plus p27 as indicated. Thirty-six hours after transfection, the samples were analyzed as described in Fig. 6A. The data represent the mean from two independent experiments.

NPAT also activates transcription from histone H2B and H3 promoters. U2OS cells were transfected with 1 μg pCMV (vector) or 1 μg pCMV–NPAT together with 50 ng pGLH2B (labeled as H2B), pGLH2B(65) [labeled as H2B(65)], pGLH2B(65M) [labeled as H2B(65M)], or pGLH3 (labeled as H3) reporter construct. 50 ng pCMV–lacZ was also cotransfected in each transfection. Twenty-two hours after transfection, cells were lysed and the fold of induction was measured as described in Fig. 6A.

Cyclin E–Cdk2 regulates NPAT-mediated activation of histone gene transcription. (A) Cyclin E–Cdk2 stimulates NPAT-mediated transcriptional activation. U2OS cells were transfected with 50 ng pGLH4(65) together with 50 ng pCMV–lacZ, and the indicated expression plasmids. 0.5 μg of each expression plasmid was used, and if needed, pCMV vector was added to bring the total amount of plasmid DNA to 1.6 μg. The samples were analyzed as described in Fig. 6A. (B) The stimulation of NPAT-mediated transcriptional activation by cyclin E–Cdk2 is the SSCS dependent. The experiments were carried out as described in A except that the reporter used was pGLH4 (M1). (C) Cyclin E–Cdk2 stimulates NPAT-mediated H4 transcriptional activation in non–S phase cells. U2Os cells, grown on 10-cm plates, were transfected with 300 ng pGLH4(65) and 300 ng pCMV–lacZ, together with indicated expression plasmids (NPAT, 6 μg; cyclin E and Cdk2, 1.5 μg). pCMV (vector) was added to bring the total plasmid DNA concentration to 9 μg/plate. Five hours after transfection, Nocadazole (final concentration 70 ng/mL) was added into the culture medium to arrest the cells in M phase. Cells in M phase were harvested by mitotic shake-off 18 h later. The cells were washed twice with culture medium and replated. One and one-half hours after replating, the effect of NPAT on H4 transcription was analyzed as described in Fig. 6A. The mean results from two independent experiments are shown. (D) Association of NPAT and cyclin E with histone gene clusters. Lysates prepared from U2OS cells were precipitated with the indicated antibodies. The indicated DNA sequences were detected by PCR using specific primers as described in Materials and Methods.