Figure 1.

H3K27me3 is a general mark for heterochromatin in Tetrahymena. (A) Schematic representation of key nuclear events in Tetrahymena conjugation. Electron-dense chromatin bodies (open arrowhead) are dispersed in the somatic macronucleus (Mac) of vegetatively growing (nonmating) cells (0 h). Two Tetrahymena cells of different mating types can pair during conjugation (only one partner is shown in immunofluorescence pictures). During this sexual pathway, the germline micronucleus (Mic) gives rise to two new micronuclei and two developing macronuclei, also referred to as anlagen (AN), supported by transcription from the parental macronuclei (PM) (6 h). As anlagen formation progresses, the old macronucleus (OM) degenerates (10 h). At late conjugation, specialized DNA elimination heterochromatic structures (solid arrowhead) form in anlagen as pairs separate (>12 h). Heterochromatic structures are highlighted (red). (B) Localization of H3K27me3. Wild-type cells were processed for immunofluorescence staining with H3K27me3-specific antibody and counterstained with DAPI. From left to right, typical examples of stained cells are shown as vegetatively growing, micronuclei/anlagen differentiation, and early and late anlagen stages, aligned with their schematic representations in A. (C) Differential usage of H3K27me3 and H3K9me3 in distinct Tetrahymena nuclei. Acid extracts from purified micronuclei and macronuclei in vegetatively growing cells (Veg) or anlagen isolated from 10-h conjugating cells (Cnj) were resolved on 10% SDS-PAGE, blotted, and probed with the indicated antibodies. Note that H3K27me3 was only observed in the fast-migrating form of micronuclear H3 (arrowhead), which corresponds to a mature, proteolytically processed H3 (Allis et al. 1980). Another further truncated form was observed in anlagen. (D) Expression of EZLs mRNA during conjugation. Total RNA samples from wild-type and ΔEZL1 cells from different conjugation time points (0, 4, 8, and 12 h post-mixing) were reverse-transcribed and analyzed by PCR with primers specific for EZL1, EZL2, EZL3, PDD1 (a conjugation-specific chromodomain protein), and HHP1 (a gene encoding a chromodomain protein ubiquitously expressed in vegetative and conjugating cells).