Bell P et al. (1997), Association of the nucleolar transcription fact...

XB-ART-15962

J Cell Sci 1997 Sep 01;110 ( Pt 17):2053-63. doi: 10.1242/jcs.110.17.2053.

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Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos.

Bell P , Mais C , McStay B , Scheer U .

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When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklace-like structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immuno-depletion experiments indicated the UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.

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Species referenced: Xenopus laevis
Genes referenced: tbp ubtf

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	Fig. 1. Immunofluorescence microscopy of Xenopus XTC-2 cells with antibodies directed against xUBF (A¢), xTBP (B¢) and pol I (C¢). All three antibodies stain the nucleoli. An additional labelling of the nucleoplasm at various intensities can be observed with the antibodies to xTBP (B¢). The corresponding phase contrast images are shown in A-C. Bar, 20 um.
	Fig. 2. Immunofluorescence microscopy of nuclei assembled from sperm chromatin in Xenopus egg extract with antibodies directed against xUBF. Besides being diffusely distributed throughout the nuclear interior, UBF is highly concentrated at one specific site per nucleus. These UBF-foci appear either as brightly fluorescent dots (A¢) or necklace-like structures consisting of tandemly arrayed fluorescent granules (B¢). The corresponding DNA-staining with Hoechst dye 33258 is shown in A and B. Bar, 10 um.
	Fig. 3. The TATA-box binding protein (TBP) is not concentrated at specific sites but rather dispersed throughout the interior of the in vitro assembled nuclei (A¢¢). The corresponding phase contrast image (A) and Hoechst fluorescence (A¢) are shown. Bar, 10 um.
	Fig. 4. UBF, pol I, and fibrillarin localize at three different sites within nuclei assembled in Xenopus egg extract. (A) Double label immunofluorescence microscopy with antibodies directed to pol I (A¢) and UBF (A¢¢) reveals that pol I is concentrated at several sites scattered throughout the nucleoplasm (A¢) but not at the UBF-positive spot (A¢¢). (B) Double label experiment with antibodies against pol I (B¢) and fibrillarin (fib; B¢¢). The pol I foci (B¢) are not identical with the nuclear bodies which contain a specific set of nucleolar proteins such as fibrillarin (B¢¢). The corresponding Hoechst fluorescence is shown in A and B. Bar, 10 um.
	Fig. 5. Nuclei assembled from sperm chromatin in UBF-deficient egg extract lack UBF-positive structures. Egg extracts were incubated with paramagnetic beads coated with either UBF antibodies or the corresponding preimmune IgGs. After retrieval of the beads, samples of the extracts and proteins bound to the beads were analyzed by immunoblotting experiments using the same antibodies. (A) UBF is detectable in control extract after incubation with preimmune IgGs (lane 1, arrow) but not in extract treated with UBF-antibodies (lane 3). Additional protein bands of 130 and 200 kDa are also stained by the UBF-antibodies on the western blot (lane 1; note, that these two proteins are not removed from the extract along with UBF, see lane 3). UBF is recovered from the beads with UBF-antibodies (lane 4, arrow) in contrast to the control beads (lane 2). The bands marked H in lanes 2 and 4 represent the heavy chain of the antibodies bound to the beads. As a control, nuclei were assembled in extract treated with preimmune IgGs. After immunostaining with UBF-antibodies these nuclei display characteristic UBF-foci (B¢). Nuclei assembled in UBF-deficient extract are completely negative with UBF antibodies (C¢). The corresponding Hoechst fluorescence is shown in B and C. Bar, 10 um.
	Fig. 6. Nuclei reconstituted from l-DNA in egg extract accumulate UBF as shown by immunofluorescence with UBF-antibodies (A¢). However, they lack the UBF-foci. DNA staining with Hoechst is shown in A. Bar, 20 um.
	Fig. 7. Nuclei assembled from sperm chromatin of wild type (A, A¢) and 1-nu Xenopus mutants (B, B¢) analyzed by immunofluorescence with UBF-antibodies. Almost all nuclei derived from sperm chromatin of wild-type (2-nu) animals reveal the characteristic UBF-focus (A¢). In contrast, about half of the nuclei assembled from sperm chromatin of 1-nu mutants lack the UBFpositive dot (B¢). The corresponding Hoechst fluorescence is shown in A and B. Bars, 10 um.
	Fig. 8. UBF colocalizes with the rRNA genes. Nuclei assembled from sperm chromatin were first stained with antibodies to UBF (A) and then subjected to in situ hybridization with a digoxigenin labelled probe complementary to part of the 28S region of Xenopus rDNA (A¢). Both procedures label the same dot-like structure. The corresponding Hoechst fluorescence is shown in A¢¢. The slightly different contours of the nuclei is a result of the hybridization procedure. Bar, 10 um.
	Fig. 9. Immunofluorescence microscopy of squash preparations of Xenopus embryos before (A¢,B¢) and after (C¢) the midblastula transition following incubation with antibodies to UBF. A dividing blastomere of a 64 cell-stage embryo in metaphase reveals two UBF-positive dots (arrows in A¢). Likewise, nuclei of an early blastula also contain two UBF-dots each (indicated by arrows; the chromatin of the two cells shown is heavily distorted due to the spreading procedure). After midblastula, the embryos eventually develop nucleoli which can be readily stained with UBF-antibodies as exemplified in a tailbud stage embryo (C¢). The corresponding Hoechst fluorescence is shown in A-C. Bars, 20 um.