Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles.

Labeling of DNA Replication and Transcription Sites and Immunofluorescence Procedures

DNA replication and transcription sites were labeled based on previous procedures ( Nakayasu and Berezney 1989 ; Jackson et al. 1993 ; Wansink et al. 1993 ) and those briefly reported by Wei et al. 1998 . Mouse 3T3 fibroblast cells are grown on coverslips in DME or MEM supplied with 10% FCS for 24–48 h. Cells are washed with ice-cold TBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 ) and further washed with glycerol buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM PMSF) for 10 min on ice. Washed cells were permeabilized with 0.025% Triton X-100 in glycerol buffer (with 25 U/ml of RNasin; Promega Corp.) on ice for 3 min and immediately incubated at room temperature for 30 min with nucleic acid synthesis buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 150 mM NaCl, 25% glycerol, 0.5 mM PMSF, 25 U/ml of RNasin, 1.8 mM ATP) supplemented with 0.5 mM CTP, GTP, and BrUTP (Sigma Chemical Co.) for labeling transcription sites (nascent RNA), 0.1 mM dATP, dCTP, dGTP, and 25 μM digoxigenin-11-dUTP (Boehringer Mannheim) for labeling DNA replication sites (nascent DNA), or both for simultaneously labeling transcription and replication sites. After incorporation, the cells were fixed with 3% freshly made paraformaldehyde in PBS on ice for 5 min, washed with ice-cold TBS-Tween buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM MgCl 2 , 0.2% Tween 20), blocked with 5% goat serum and incubated with rat anti-BrU antibodies (IgG, SeraLab) followed by biotin-conjugated goat anti–rat IgG (1:50; Jackson ImmunoResearch Laboratories, Inc.) and Texas red–conjugated streptavidin (1:100; GIBCO BRL) to detect transcription sites. Replication sites were detected with FITC-conjugated sheep antidigoxigenin Fab fragments (1:10; Boehringer Mannheim). All incubations were performed at room temperature for 30 min. Alternatively, nascent RNA was labeled in vivo, by pulsing 3T3 cells for 2 min with 30 μM BrU (Sigma Chemical Co.). The biotin-strepavidin enhancement system resulted in greatly improved sensitivity for detected individual transcription sites compared with a standard secondary antibody approach and was absolutely essential for detecting transcription sites after 2-min in vivo pulses of BrU. Nuclear speckles were decorated with the Y12 mAb to Sm, which recognizes common core proteins of snRNPs involved in RNA processing ( Lerner et al. 1981 ; Zieve and Sauterer 1990 ), followed by goat anti–mouse IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.) conjugated to FITC or Texas red. Coiled bodies and the nuclear lamina were decorated with rabbit polyclonal anticoilin and antilamin B antibodies, respectively, and detected with FITC- or Texas red–conjugated goat anti–rabbit IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted on slides in Slow-Fade (Molecular Probes, Inc.) and stored at −20°C for microscopic examination. Laser Scanning Confocal Microscopy, Image Processing, and Quantitation Optical sections (0.5 or 0.3 μm where indicated) were collected with a confocal microscope (MRC-1024; Bio-Rad) equipped with a Nikon Optiphot 2 microscope, a Nikon 60×, 1.4 NA objective, and a krypton argon laser to simultaneously excite FITC and Texas red at 488 and 568 nm, respectively. Emissions were collected with filters 522-DF32 for FITC and HQ598-40 for Texas red. The pixel intensity distribution was checked before image collection and adjusted so that the pixel intensities were all below saturation. The total number of transcription sites are calculated by a new and highly improved segmentation program, presented in detail elsewhere ( Samarabandu et al. 1995 ). In brief, by using two-dimensional image segmentation on the individual slices to obtain transcription site contours and combining this high level two-dimensional data using a modified three-dimensional connected component labeling algorithm with weak connectivity in the Z direction, we were able to reconstruct the network of three-dimensional transcription sites. Instead of using the equivalent of traditional two-dimensional 8-connectivity in three dimensions, we used a metric that evaluated the amount of overlap between successive sections to determine whether the two contours belong to the same site. After determining the three-dimensional boundary of transcription sites, the center of gravity of each site was calculated by averaging the (x, y, z) coordinates of all voxels that belong to the site ( Samarabandu et al. 1995 ). Segmentation of the nuclear speckles, nucleoli, the total nuclei, and measurement of the size and intensity of segregated areas was performed in IPLab (Signal Analytics Corp.). Distribution of transcription sites between the nuclear speckles and other nuclear regions was determined by direct measurement. Fluorescence signal intensities from unprocessed confocal microscopic images were measured in IPLab. In brief, the extranucleolar regions, nucleolar regions, and the internal region of the nuclear speckles were manually selected and the mean intensities were calculated after multiple scans through the areas under analysis. The nucleolar signal was selected as background and subtracted from the signal emitting from the speckle and extranucleolar regions.

Labeling of DNA Replication and Transcription Sites and Immunofluorescence Procedures

DNA replication and transcription sites were labeled based on previous procedures ( Nakayasu and Berezney 1989 ; Jackson et al. 1993 ; Wansink et al. 1993 ) and those briefly reported by Wei et al. 1998 . Mouse 3T3 fibroblast cells are grown on coverslips in DME or MEM supplied with 10% FCS for 24–48 h. Cells are washed with ice-cold TBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 ) and further washed with glycerol buffer (20 mM Tris-HCl, pH 7.4, 25% glycerol, 5 mM MgCl 2 , 0.5 mM EGTA, 0.5 mM PMSF) for 10 min on ice. Washed cells were permeabilized with 0.025% Triton X-100 in glycerol buffer (with 25 U/ml of RNasin; Promega Corp.) on ice for 3 min and immediately incubated at room temperature for 30 min with nucleic acid synthesis buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 150 mM NaCl, 25% glycerol, 0.5 mM PMSF, 25 U/ml of RNasin, 1.8 mM ATP) supplemented with 0.5 mM CTP, GTP, and BrUTP (Sigma Chemical Co.) for labeling transcription sites (nascent RNA), 0.1 mM dATP, dCTP, dGTP, and 25 μM digoxigenin-11-dUTP (Boehringer Mannheim) for labeling DNA replication sites (nascent DNA), or both for simultaneously labeling transcription and replication sites. After incorporation, the cells were fixed with 3% freshly made paraformaldehyde in PBS on ice for 5 min, washed with ice-cold TBS-Tween buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM MgCl 2 , 0.2% Tween 20), blocked with 5% goat serum and incubated with rat anti-BrU antibodies (IgG, SeraLab) followed by biotin-conjugated goat anti–rat IgG (1:50; Jackson ImmunoResearch Laboratories, Inc.) and Texas red–conjugated streptavidin (1:100; GIBCO BRL) to detect transcription sites. Replication sites were detected with FITC-conjugated sheep antidigoxigenin Fab fragments (1:10; Boehringer Mannheim). All incubations were performed at room temperature for 30 min. Alternatively, nascent RNA was labeled in vivo, by pulsing 3T3 cells for 2 min with 30 μM BrU (Sigma Chemical Co.). The biotin-strepavidin enhancement system resulted in greatly improved sensitivity for detected individual transcription sites compared with a standard secondary antibody approach and was absolutely essential for detecting transcription sites after 2-min in vivo pulses of BrU. Nuclear speckles were decorated with the Y12 mAb to Sm, which recognizes common core proteins of snRNPs involved in RNA processing ( Lerner et al. 1981 ; Zieve and Sauterer 1990 ), followed by goat anti–mouse IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.) conjugated to FITC or Texas red. Coiled bodies and the nuclear lamina were decorated with rabbit polyclonal anticoilin and antilamin B antibodies, respectively, and detected with FITC- or Texas red–conjugated goat anti–rabbit IgG (1:20–50; Jackson ImmunoResearch Laboratories, Inc.). Coverslips were mounted on slides in Slow-Fade (Molecular Probes, Inc.) and stored at −20°C for microscopic examination.