BXB-ER activation induces changes in H3K9Me3/HP1 distribution between chromocentres and nucleoplasm and spreads H3K9Me3 to telomeres.

<p>(A) Optical sections of cells that were immunoprocessed for both H3K9Me3 (red in double stains) and HP1β (green in double stains) as indicated above the panels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013322#pone.0013322-Martin2" target="_blank">[77]</a>. The staining pattern reveal the spreading of H3K9Me3 and HP1β to numerous nucleoplasmic sites upon BXB-ER activation. Normal chromocentres with co-localized H3K9Me3/HP1β appear yellow (arrowheads) and chromocentres with reduced HP1® appear red (arrows). Individual immunoflorescences are shown in grey. Scale bars: 20 µm. (B/C) Proportions of cells with immunofluorescence restricted to chromocentres versus cells with additional nucleoplasmic signals were scored (223 and 129 cells for HP1β and H3K9Me3, respectively). (D/E) Metaphase spreads from indicated cells, after growing without or with estrogen (*) for 15 hours, were immunoprocessed for H3K9Me3 (red) and DNA was counterstained with Sytox (green). Optical slices (D) and corresponding 3D reconstructions (E <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013322#pone.0013322-Martin1" target="_blank">[38]</a>) show normal pericentric H3K9Me3 staining (arrow) and new distinct, small sites of H3K9Me3 on chromosome arms and telomeres (arrowheads) after activation. Scale bars: 5 µm. (F) Control experiment to show that estrogen treatment has no influence on the pattern of H3K9Me3 distribution in interphase or mitotic 3T3 cells. Untreated 3T3 cells or 3T3 cells treated for 15 hours with estrogen (*) were immunoprocessed for H3K9Me3 (red) and DNA was counterstained with Sytox (green). Optical slices of fields showing metaphase spreads (M) and interphase cells (I) are shown. Scale bars: 10 µm.