Corsetti MT et al. (1995), Nucleolar localisation of three Hox homeoproteins.

XB-ART-20351

J Cell Sci 1995 Jan 01;108 ( Pt 1):187-93.

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Nucleolar localisation of three Hox homeoproteins.

Corsetti MT , Levi G , Lancia F , Sanseverino L , Ferrini S , Boncinelli E , Corte G .

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Homeoproteins encoded by genes of the Hox family are nuclear proteins believed to act as transcription factors and to participate in the determination of the body plan. Here we show that in several vertebrate cells, they exhibit a subnuclear localisation associated with the nucleolus. We used monoclonal antibodies to study the distribution of three homeoproteins, namely HOXB7, HOXC6 and HOXD4. The immunoreactivity to antibodies against HOXC6 protein in Xenopus laevis embryonic tissues is restricted to one or two spots within the nucleus; this distribution partially overlaps that of fibrillarin, a protein of the fibrillar zone of the nucleoli. Indirect immunofluorescence analysis of the distribution of HOXB7 protein in 3T3 cells, and of HOXD4 protein in human neuroblastoma and Raji lymphoma cell lines and activated lymphocytes, results invariably in a nucleolar localisation. Purified nucleoli from stimulated T lymphocytes, and Raji cells contain an activity capable of binding, in a gel retardation assay, to an oligonucleotide specifically recognised by the HOXD4 homeoprotein. This activity is specifically removed by anti-HOXD4 antibodies and is found associated in southwestern blots with a single band with an apparent M(r) of 30,000, corresponding to that of recombinant HOXD4. The functional significance of the nucleolar localisation of Hox proteins remains to be determined.

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Species referenced: Xenopus laevis
Genes referenced: hoxb7 hoxc6 hoxd4

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	Fig. 1. Distribution of 3B2 (anti-HOXC6) immunoreactivity in developing Xenopus tissues. Strong staining is always localised within the nucleolus. (A) Confocal microscope analysis of a section through a rostral region of the spinal cord of a stage 45 embryo. Double staining with 3B2 monoclonal antibody (green) and a polyclonal anti-NCAM antibody (red). Most neuronal cell bodies are positive for both molecules. While N-CAM stains all neuronal plasma membranes, anti-HOXC6 immunoreactivity is localised in one or two spots within the nucleus. ep, ependyma. (B) Section through the visceral part of a stage 41 embryo, double stained with propidium iodide and 3B2 monoclonal antibody. All nuclei are stained in orange. Nuclei of the intestinal epithelium contain a bright yellow spot corresponding to the stain of 3B2 monoclonal antibody. Nuclei of the overlying epidermids (e) are not stained by 3B2 antibody. (C-E) High magnification confocal analysis of sections through the nervous system (C,D) and through the liver (E). Double staining with 3B2 monoclonal antibody (green) and anti-fibrillarin antibody (Lapeyere et al., 1990) (red). Nucleoli of neural cells are stained by both antibodies, whereas nucleoli from liver are only stained by anti-fibrillarin antibodies. A,B, ´400; C,D, ´5000; E, ´2000.
	Fig. 2. Staining of human and mouse cells with 5BL (anti-HOXB7), 3B2 (anti-HOXC6) and 4C5 (anti-HOXD4) monoclonal antibodies. (A) Mouse 3T3-J2 cells stained with 5BL. (B) Mouse 3T3-J2 cells stained with 3B2. (C) Mouse 3T3-J2 cells stained with 4C5. (D) Raji cells stained with 5BL. (E) Raji cells stained with 3B2. (F) Raji cells stained with 4C5.
	Fig. 3. Staining of human cells with 5BL (anti-HOXB7) and 4C5 (anti-HOXD4) monoclonal antibodies. (A) SK-N-SH human neuroblastoma cells before induction with retinoic acid, stained with 4C5. (B) SK-N-SH human neuroblastoma cells induced with retinoic acid, stained with 4C5. (C) Human resting peripheral blood lymphocytes stained with 4C5. (D) Human peripheral blood T cells 24 hours after stimulation with phytohemoagglutinin (PHA), stained with 4C5. (E) Human peripheral blood T cells 72 hours after stimulation with PHA, stained with 4C5. (F) Human peripheral blood T cells 72 hours after stimulation with PHA, stained with 5BL. ´630.
	Fig. 4. A band-shift experiment showing copurification with the nucleoli of Raji cells and activated T lymphocytes of a protein binding to a oligonucleotide recognised by the HOXD4 homeoprotein. (A) Lane 1, total nuclear extract from Raji cells; lane 2, extract from purified nucleoli from Raji cells; (B) lane 1, nucleolar extract from activated T cells; lane 2, nucleolar extract from resting peripheral blood T cells. B. The 4C5 monoclonal antibody specifically removes the binding protein from nucleolar extracts of Raji cells. (C) Lane 1, nucleolar extract incubated with Sepharosebound human Igs; lane 2, nucleolar extract incubated with Sepharose-bound 4C5 antibody; lane 3, no extract.
	Fig. 5. In southwestern blots of extracts obtained from nucleoli positive with the 4C5 antibody a single band with an apparent Mr of 30,000 binds to the 4B-26 oligonucleotide. (A) A typical preparation of purified nucleoli stained with Toluidine Blue. ´1200. (B) Nucleolar extract from Raji cells and resting T lymphocytes were subjected to SDS-PAGE in a 12% polyacrylamide gel, in duplicate. After electrophoresis half of the gel was silver stained and the other half transferred to nitrocellulose according to standard procedures. The blot was then denaturated/renaturated, incubated with the radioactive oligonucleotide and washed as previously described. Lanes 1 and 2, silver staining of nucleoli from Raji cells and T lymphocytes, respectively; lanes 3 and 4, autoradiography of the corresponding southwestern blot.