Figure 6. A rapid screen of mosaic analysis identifies histone modification sites involved in polycomb-group repressin.

(A) Pattern of cell division in epidermal cells from flies with 20 His-GUs (20 copies of wild-type histone gene units) and H3K27A mutations. Panels show time-matched individual embryos labeled for Cyclin B (white), H3K27me3 antibody (red), and DAPI (blue). Scale bar: 100 µm.

(B) Clonal analysis demonstrates H3K27 is required for polycomb target-gene repression. Wing imaginal disc clones of His^D homozygous cells in animals with transgenic His-GUs^WT (left) or His-GUs^H3K27A (right) were immunostained with anti-Ubx and anti-H3K27me3. His^D homozygous cells with transgene are marked by the absence of green fluorescent protein (GFP). The yellow box shows the clone cells. Scale bar: 50 µm.

(C) H3R26 is essential for efficient polycomb-mediated gene repression in Drosophila. Wing imaginal discs clones of His^D homozygous cells in animals with transgenic His-GUs^H3R26A were immunostained with anti-Ubx or anti-Abd-B, anti-H3K27me3 or anti-H3K9me3. His^D homozygous cells with His-GUs^H3R26A are marked by the absence of green fluorescent protein (GFP). Both Ubx and Abd-B were mis-expressed in homozygous H3R26A mutant cells (left column) and H3K27me3 was strongly reduced (top right corner). By contrast, H3K9me3 was unaffected in H3R26A clones (lower right corner).

(D) H3K27 tri-methylation is decreased in H3R26A Drosophila embryos. Western blot assay on lysate from 20 His-GUs, H3K27A, H3R26A, H3S28A, and H3K9A flies immunoblotted using anti-H3K27me3 antibody.

(E) Quantification of Figure 6D western blots. Densitometry of western blot bands was quantified with Image J software. Values are means ± SEM of three biological replicates.

See also Figure S7.