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First published online 5 May 2004
doi: 10.1242/jcs.01238

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Heterochromatin and tri-methylated lysine 20 of histone H4 in animals

¹ Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, Roslin Institute, Edinburgh, EH25 9PS, UK
² Laboratory of Developmental Genetics, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK
³ Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA
⁴ Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, Via San C. De Lellis, Viterbo, 01100, Italy
⁵ Dipartimento di Genetica e Biologia molecolare, Università di Roma `La Sapienza', Piazzale Aldo Moro, Roma, 00185, Italy
⁶ Uppsala University, Department of Development and Genetics, Norbyvagen 18A, Uppsala, 75236, Sweden

View larger version (72K): [in a new window]   Fig. 1. Specificity of the antibody for tri-methylated lysine 20 of histone H4 (anti-Me(3)K20H4). (A) The anti-Me(3)K20H4 antibody recognises methylated H4 by western blotting. Histones from various sources were separated by SDS-PAGE, blotted and probed with anti-Me(3)K20H4. The sources of the histones were: turkey erythrocyte nuclei (lane 1), turkey erythrocyte core histones (lane 2), recombinant H4 (lane 3) and calf thymus core histones (lane 4). The bottom row shows a Coomassie Blue-stained profile of the samples used in the immunoblot. (B) Specificity of anti-Me(3)K20H4 antibody for the trimethylated form of K20 as determined by dot blotting. Serial dilutions (lanes left to right) of the non modified (I), mono- (II), di- (III) and tri-methylated (IV) K20 peptides were dot blotted onto nitrocellulose and then probed with anti-Me(3)K20H4. (C) The antibody is specific for Me(3)K20H4 in a competition assay using specific methylated peptides and membrane-bound histone H4. Calf thymus histone H4 is identified on an immunoblot by the anti-Me(3)K20H4 antibody alone (lane 1) or preincubated with non modified (lane 2), Me(1)K20H4 peptide (lane 3) and Me(2)K20H4 peptide (lane 4). Preincubation of the antibody with the Me(3)K20H4 peptide abolishes binding to H4 histone (lane 5). (D) The antibody is specific for Me(3)K20H4 in a competition assay using immunofluorescence (bottom row; top row are the corresponding DAPI profiles). From left to right: no competitor, non modified K20H4 peptide, Me(1)K20H4 peptide, Me(2)K20H4 peptide, Me(3)K20H4 peptide. In the last panel a Me(3)K9H3 was used as a competitor. Only the Me(3)K20H4 peptide competes. Scale bar: 5 µm.

View larger version (18K): [in a new window]   Fig. 2. Distribution of Me(3)K20H4 in an asynchronous population of C127 mouse cells. (A) The DAPI profile of C127 cells. (B) The same field as in A stained for the Me(3)K20H4. (C) The merged image of A and B; DAPI is pseudocoloured in red and Me(3)K20H4 in green. Scale bar: 5 µm.

View larger version (109K): [in a new window]   Fig. 3. Distribution of Me(3)K20H4 in murine metaphase chromosomes. (A) DAPI profile of the chromosome-spread from a mouse C127 cell. (B) The spread in A labelled with anti-Me(3)K20H4, which shows labelling of peri-centromeric heterochromatin. The labelling is punctate, as seen in the inset, which is a magnified image of the centromeric Me(3)K20H4 staining indicated by the asterisk. (C) The spread in A labelled with anti-AcH4 antibody. (D) The merged image of B and C. Arrows in B,C and D point to the inactive X chromosome (Xi), which is underacetylated in C. Scale bar: 5 µm.

View larger version (35K): [in a new window]   Fig. 4. Me(3)K20H4 replicates late in S-phase of the cell cycle. C127 cells were arrested at the G1/S border with mimosine and then released into S-phase. Cells were pulse labelled with BrdU either early (2 hours after release, S-E) or late (8 hours after release; S-L) in S-phase, and were then stained for DAPI (blue), BrdU (red) and Me(3)K20H4 (green). In the far right column, the BrdU and Me(3)K20H4 staining patterns were simultaneously visualised through a dual FITC/Texas Red filter (Dual Filter). This showed that the BrdU and Me(3)K20H4 staining patterns colocalise late in S-phase (S-L). Scale bar: 5 µm.

View larger version (40K): [in a new window]   Fig. 5. Decoration of DAPI-positive heterochromatin blocks with anti-Me(3)K20H4 antibody in C127 cells is enhanced upon serum removal. C127 cells were cultured in normal culture medium (A,B) or in culture medium containing 0.4% (C,D) and 0.1% (E,F) foetal calf serum. (A,C,E,G) Staining with anti-Me(3)K20H4; (B,D,F,H) DAPI profiles of the same fields. One heavily decorated nucleus, typically found in cells cultured in low serum concentration, is shown in G and H. Scale bars: 10 µm (A-F); 5 µm (G,H)

View larger version (88K): [in a new window]   Fig. 6. Me(3)K20H4 localises to the centromeric and telomeric heterochromatin during spermatogenesis and transiently accumulates at the PAR region. Me(3)K20H4 distribution (green) is followed in pachytene and diplotene nuclei. The axial elements of the paired chromosomes are visualised with anti-SYCP3 antibodies (red) and the centromeres with human anti-centromeric antibodies (ACA; blue). During early (A-C) and mid-pachytene (D-F), Me(3)K20H4 localises at the centromeres, some of the telomeres (arrows in C and F) and the PAR region of the XY-body. The centromeric heterochromatin of one of the autosomes (asterisk) and of the Y chromosome are not stained with anti Me(3)K20H4 antibodies. At late pachytene (G-I) and diplotene (J-L) the PAR (arrow indicates the PAR) and telomeric staining of Me(3)K20H4 have disappeared.

View larger version (29K): [in a new window]   Fig. 7. Me(3)K20H4 is present in mature sperm. Hypotonically swollen sperm stained with DAPI (A) and anti-Me(3)K20H4 antibodies (B). (C) Merged image of A and B. Me(3)K20H4 staining colocalises with the bright (AT-rich) DAPI staining blocks. Me(3)K20H4 staining of the sperm is weak; when compared to the single spermatid, below the sperm cells (the `double-dot' staining), the intensity of the Me(3)K20H4 staining in the sperm cells is weak, but detectable. Scale bar: 5 µm.

View larger version (68K): [in a new window]   Fig. 8. Distribution of Me(3)K20H4 in oocytes and one- and two-cell mouse embryos. (A,D,G,J,M,P) Oocytes and one- and two-cell embryos stained with DAPI. The sperm (sp), maternal (ma), paternal (pa) and polar body (pb) DNA stains intensely with DAPI compared with the cytoplasm. (B,E,H,K,N,Q) The same embryos were stained using the anti-Me(3)K20H4 antibody and the immunofluorescence (FITC) is shown in green. (C,F,I,L,O,R) Merged images of the embryos in the left and the middle columns. DAPI is pseudocoloured in red; colocalisation is yellow. (A-C) Me(3)K20H4 is enriched in the maternal centromeres of a metaphase II-arrested oocyte. (D-F) Approximately 1 hour after fertilisation (n=10) the sperm nucleus is found in the ooplasm and lacks Me(3)K20H4, while the polar body and the maternal chromosomes contain Me(3)K20H4. (G-I) Me(3)K20H4 is present in the maternal pronucleus but undetectable in the paternal pronucleus (pronuclear stage: n=32). (J-L) At syngamy (n=33) the difference in Me(3)K20H4 between the parental chromosomes is clear. (M-O) Me(3)K20H4 staining is partitioned in the late telophase/early G1 phase two-cell embryo (early two cell; n=15). (P-R) At late two-cell embryos (n=35) the intensity of Me(3)k20H4 staining is very weak. Scale bars: 10 µm (A-L); 10 µm (M-R).

View larger version (46K): [in a new window]   Fig. 9. Me(3)K20H4 distribution on Drosophila polytene chromosomes. A polytene chromosome spread stained with DAPI (A) and anti-Me(3)K20H4 (B). (C) Merged image of A and B. Me(3)K20H4 has a punctate appearance and is concentrated at the chromocenter (arrow) and at numerous euchromatic bands.

View larger version (21K): [in a new window]   Fig. 10. Me(3)K20H4 is present only at the paternal chromosomes of male mealy bugs. Prometaphase male mealy bug cells stained with DAPI (A) and anti-Me(3)K20H4 (B). (C) Merged image of A and B. Me(3)K20H4 (green) has a punctate appearance and is distributed through the paternal chromosome set, which stains intensely with DAPI (pseudocoloured in red). Scale bar: 5 µm.

View larger version (74K): [in a new window]   Fig. 11. Distribution of Me(3)K20H4 is dependent upon Suv 3(9)h1/h2 H3-specific HMTase activity. B is the merged image of A and C, and F is the merged image of E and G; DAPI is pseudocoloured red and Me(3)K20H4 green. Staining of Suv3-9 null cells with the anti-Me(3)K20H4 antibody shows that Me(3)K20H4 is distributed throughout the nucleoplasm (C), outside the DAPI-positive blocks of constitutive heterochromatin (A; merged image in B). In a mitotic nucleus from the Suv3-9 null cell line the Me(3)K20H4 is absent from centromeric heterochromatin (D). This is in stark contrast to a mitotic cell from wild-type mouse fibroblasts (H), where there is robust staining of the centromeric heterochromatin. In an asynchronous population of wild type cells, the staining of Me(3)K20H4 is variable (G), as previously described (see Figs 2 and 5), and present over the DAPI-positive blocks of heterochromatin (E; merged image in F).