Preferential distribution of active RNA polymerase II molecules in the nuclear periphery

Abstract

We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.

Figure 1

Immunoelectron microscopic localization technique for whole mount nuclei. Diagrammatic representation of the steps employed in the immunoelectron microscopic localization of nuclear antigens. Nuclei are reacted with primary antibody in suspension, deposited on EM grids by centrifugation, washed, and reacted with secondary antibody conjugated to colloidal gold particles. A nuclear protein is immunolocalized in situ by the visualization of colloidal gold particles under the EM.

Figure 2

Immunoelectron microscopic labeling of control antigens. The reaction of a anti-biotin on mouse L929 cell nuclei, b anti-histone H1A on HeLa cell nuclei, and c anti-topoisomerase I on mouse L929 cell nuclei, followed by immunogold labeling (15 nm particles) as described in Methods, a. Rabbit anti-biotin antibody was used at a dilution of 1:50. b. Antibodies against human histone subtype HIA were obtained by injection of purified human placental histone H1A into rabbits by a modification of the Vaitukaitis method (Lewis et al., 1977). IgGs were purified by Affigel-blue (Bio-Rad) chromatography and concentration by ultrafiltration. A dilution of 1:50 was used for immunoelectron microscopic localization, c. Anti-topoisomerase I antibodies (the kind gift of Dr. Gerd Maul) were derived from a human scleroderma patient and purified by Affigel-blue (Bio-Rad) chromatography followed by ultrafiltration. A dilution of 1:20 to 1–50 was used for immunoelectron microscopic localization. Colloidal gold-secondary antibody complexes define antigen distributions. A single representative nucleus is shown in each case. Bar: 1 µm.

Figure 3

Electrophoretic analysis and immunodetection of the large subunit of RNA polymerase II. Whole cell extracts (40 µg of protein, lane 1), nuclear extracts (20 µg of protein, lane 2), or purified calf thymus RNA polymerase II (0.1 µg of protein, lane 3) were separated on 10% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose as described by Towbin et al. (1979) and reacted with affinity-purified anti-fusion protein antibody, followed by iodinated goat anti-rabbit IgG. An autoradiogram of the immunoreactive proteins is shown.

Figure 4

Immunoelectron microscopic localization of RNA polymerase II. Mouse L929 cell nuclei were reacted with one of two antibodies prepared against RNA polymerase II. Antibodies used in a were directed against a fusion protein containing a portion of β-galactosidase and the fifth exon of human RNA polymerase II (Cho et al., 1985; Rappaport et al., 1988). IgGs were prepared by ammonium sulfate precipitation and DE52 chromatography followed by a β-galactosidase affinity column to remove antibodies against the bacterial protein. A dilution of 1:40 was used. The antibodies used in b were prepared against total calf thymus RNA polymerase II (Kim and Dahmus, 1986) and were the generous gift of Dr. Michael Dahmus. They were purified by Affigel-blue (Bio-Rad) chromatography, concentrated by ultrafiltration, and used at a dilution of 1:40. In both cases, reaction sites were located with goat anti-rabbit colloidal gold. Bar: 1 μm.

Figure 5

Immunoelectron microscopic localization of lamins. Mouse L929 cell nuclei were reacted with anti-lamin antibodies and secondary antibodies on colloidal gold. Antibodies derived from human autoimmune sera (McKeon et al., 1983) were the kind gift of Dr. Marc Kirschner. IgGs were purified by Affigel-blue (Bio-Rad) chromatography and concentrated by ultrafiltration. A dilution of 1:200 was used for immunoelectron microscopic localization. Bar: 1 μm.

Figure 6

Immunolocalization of RNA polymerase II after α-amanitin treatment of cells. Mouse L929 cells were incubated in 5 μg/ml α-amanitin for 4 hours, and nuclei were released and incubated with anti-RNA polymerase II, followed by immunogold labeling as described in Methods. Bar: 1 μm.

Figure 7

Immunolocalization of sites of transcription in a whole mount nucleus. Isolated nuclei were incubated under run-off transcription conditions (Weber et al., 1977) in the presence of biotinylated UTP in order to label sites active in transcription. Incorporated nucleotides were located with streptavidin-gold. Bar: 1 μm.