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. 2014 Mar 20;10(3):e1004240. doi: 10.1371/journal.pgen.1004240

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© 2014 Satyaki et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) mel-Hmr-HA (green) colocalizes with HP1a (top) and H3k9me2 (middle; both red) in nuclear cycle 14 embryos. The HP1a costain is in a mel-Hmr-HA background, while the H3k9me2 costain is in a Hmr³; mel-Hmr-HA background. A negative control shows no HA signal in w¹¹¹⁸ embryos lacking the mel-Hmr-HA transgene (bottom). Scale bars represent 10 µm. (B) mel-Hmr-HA (green) colocalizes with 2L3L, dodeca and GA-rich satellites but not with the 359 bp repeat satellite in mel-Hmr-HA (all FISH probes red). Scale bars represent 5 µm. (C) mel-Hmr-HA (red) colocalizes with the nucleolar marker Fibrillarin (green) in mel-Hmr-HA early embryos. Scale bars represent 10 µm. (D) mel-Lhr-HA and mel-Hmr-FLAG co-immunoprecipitate from D. melanogaster embryo extracts derived from flies expressing both transgenes (left 4 lanes) but not from flies expressing only Lhr-HA (right 4 lanes). Extracts were IP'd with the indicated antibodies, and then probed by Western Blots (WB) with the same or different antibodies. (E) Lhr-HA, Hmr-FLAG and HP1a co-immunoprecipitation from embryo extracts. Specificity is indicated by lack of immunoprecipitation of histone H3. Asterisk indicates the antibody light chain. (F) Lhr and Hmr interact in a yeast-two hybrid assay. Interactions were detected by growth on complete media (CM) lacking histidine (his); growth controls were performed on CM lacking tryptophan (trp) and leucine (leu). The top 4 panels test for interactions between orthologs from the same species; the bottom 4 between heterospecific orthologs. AD, activation domain; BD, DNA binding domain. (G) Lhr-HA is detectable in Hmr³ and localizes to heterochromatin, as indicated by co-localization with HP1a. Note that a higher gain was used in the Hmr³ panels compared to the Hmr⁺ panels in order to detect Lhr-HA, and is reflected in the higher background. Western blots confirm that Lhr-HA levels are reduced in Hmr³. HP1a is used as a loading control. (H) Hmr-HA maintains its localization to DAPI-dense heterochromatin in Lhr^KO; Hmr-HA embryos. Scale bars represent 10 µm.