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First published online 27 February 2007
doi: 10.1242/jcs.03412

Journal of Cell Science 120, 1072-1080 (2007)
Published by The Company of Biologists 2007
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Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway

Silvia Bongiorni1, Barbara Pasqualini1, Monia Taranta1, Prim B. Singh2 and Giorgio Prantera1,*

1 Department of Agrobiologia e Agrochimica, University of Tuscia, 01100 Viterbo, Italy
2 Division of Tumor Biology, Dept. of Immunology and Cell Biology, Forschungszentrum Borstel, 23845 Borstel, Germany


Figure 1
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  		Fig. 1. Localisation of C1A9 and Me(3)K9H3 antibodies to nuclei in mid-cleavage embryos (128-256-nuclei embryos) undergoing facultative heterochromatinisation. (A) Embryo region where the wave of facultative heterochromatinisation is spreading from the bottom left corner to the top right corner. Boxed area B shows nuclei that have completed heterochromatinisaton and contain DAPI-positive chromocenters (see magnified image in B), whereas boxed area C shows nuclei still undergoing heterochromatinisation, many of which have no overt DAPI-positive chromocenters (see arrows pointing to nuclei in C). The nuclei in B are labeled with C1A9 antibody (B') and with the anti-Me(3)K9H3 antiserum (B") and the merged image in B'" shows colocalisation of DAPI-positive chromocenter with C1A9 and Me(3)K9H3 staining. The DAPI-stained nuclei in C were stained with C1A9 antibody (C') and with the anti-Me(3)K9H3 antiserum (C"). Although the pattern of Me(3)K9H3 staining is more spread out in these nuclei compared with those that have completed heterochromatinisation (see C" and B") the merged image in C'" shows that the Me(3)H9H3 pattern largely colocalises with C1A9 staining. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. C1A9 and Me(3)K9H3 staining in nuclei of cells from a tract of a male gut undergoing developmental reversal of heterochromatinisation. (A) Dissected gut of a male mealybug. (B) DAPI staining of nuclei, showing that the chromocenters of nuclei undergoing reversal of heterochromatinisation are decondensed and have lost their focal morphological appearance. (C,D) C1A9 staining of these nuclei is grainy and dispersed over the nucleus (C), whereas Me(3)K9H3 is still concentrated over the decondensed chromocenter (D). (E) Merged image. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Localisation of Me(3)K20H4 to nuclei either undergoing facultative heterochromatization or developmental de-heterochromatinisation. (A) DAPI-stained nuclei from a mid-cleavage stage embryo (128-256-nuclei embryo) that has undergone facultative heterchromatinisation. A clear, strongly-staining, chromocenter can be seen in each nucleus. The nuclei shown in A were labeled with C1A9 antibody (A') and with the anti-Me(3)K20H4 antiserum (A"); the merged image in A'" shows co-incidence of DAPI-positive chromocenters with C1A9 and Me(3)K20H4 staining. The nuclei in B are from another region of the same embryo that has yet to complete heterochromatinisation and several of them have no overt DAPI positive chromocenters. The DAPI-stained nuclei in B were stained with C1A9 antibody (B') and with the anti-Me(3)K20H4 antiserum (B"). Whereas the Me(3)K20H4 staining is more spread out in these nuclei compared with those that have completed heterochromatinisation (see B" and A") the merged image (B'") shows that the Me(3)H20H4 pattern largely colocalised with C1A9 staining. (C) DAPI-stained nuclei taken from cells of adult tissues that undergo developmental reversal of heterochromtinisation. C1A9 staining is dispersed and has a grainy appearance over the nucleus (C'). There is no staining of Me(3)K20H4 in the nucleus; rather, Me(3)K20H4 is found in the cytoplasm (C"). (C'") Merged image. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Mid-cleavage male embryos treated for 4 hours with pchet2 dsRNA exhibit a dramatic loss in facultative heterochromatinisation of the paternal chromosome set. (A) DAPI staining of a 128-256-nuclei male embryo treated for 4 hours with pchet2 dsRNA showing a loss of strongly staining DAPI-positive chromocenters. These male embryos can only be distinguished from female embryos because of male-specific chromocenter remnants (arrow in A'). (A") C1A9 staining is not detectable in these embryos. (B-B") In control mock-treated embryos typical, bright, DAPI-positive chromocenters can be seen (B and B') that are also positive for C1A9 staining (merged image in B"); DAPI staining (red), C1A9 staining (green). Bars, 10 µm.

 

Figure 5
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  		Fig. 5. pchet2 dsRNA has an effect after 2 hours and mitotic chromosomes are particularly sensitive to the treatment. (A) DAPI staining of a 128-256-nuclei male embryo treated for 2 hours showing a decondensing chromocenter and detectable C1A9-staining that is becoming dispersed and more grainy in appearance (A'). Mitotic chromosomes show a marked reduction in C1A9 staining. (B,B') Both mitotic chromosomes and an interphase nucleus are stained with DAPI and C1A9, respectively. In B' the mitotic chromosomes are not stained with C1A9, whereas the interphase nuclei still possess a C1A9-positive chromocenter (arrow in B'). After 4 hours the chromocenters have been almost completely disassembled, with only remnants remaining (arrows in C and D). The remnants did not stain with C1A9 (C',D') or Me(3)K9H3 (C"), or with Me(3)K20H4 (D"). Bars, 10 µm.

 

Figure 6
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  		Fig. 6. pchet2-dsRNA-treated embryos of both sexes exhibit defects in chromosome condensation and integrity as well as segregation defects. (A-E) DAPI-stained nuclei from pchet2-dsRNA-treated 128-256-stage embryos often possess highly elongated chromosomes that result from incomplete condensation. Mitotic figures also contain `dot' chromosomes (arrows in B and C). pchet2-RNA-treated embryos exhibit segregation defects, showing chromosomes that are displaced from the metaphase plate (arrows in D and E). For comparison, a normal metaphase from mocktreated embryos is shown in F. Bar, 10 µm.

 

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