Figure 1. EGO-1 Activity Is Required for H3K9me2 Accumulation on Unpaired Chromosomes and Chromosomal Fragments during Meiosis.

Each panel shows meiotic nuclei stained with DAPI to visualize DNA or polyclonal anti-H3K9me2 antibody. ego-1 null alleles are used in all cases (see Supplemental Experimental Procedures). Photographs are oriented with distal tissue to the left.

(A) Wild-type (wt) male pachytene cells contain a bright focus of H3K9me2 staining associated with the condensed X chromosome. Weaker staining is visible on autosomes. H3K9me2 staining rapidly decreases as cells exit pachytene and start to condense as primary spermatocytes (also see Figure S1). In ego-1 mutant males, the condensed X chromosome is distinct in DAPI-stained preparations but fails to accumulate a bright focus of H3K9me2 staining (arrows). This result is consistent with previous work showing that condensation of the male X can occur in the absence of H3K9me2 enrichment [1]. Weak H3K9me2 staining is visible on all chromosomes. We note that occasional ego-1 nuclei had one or more brighter foci of staining that did not seem to localize to the X, suggesting that EGO-1 activity may also prevent inappropriate H3K9me2 accumulation on autosomes.

(B) him-8 and ego-1/+;him-8 pachytene cells have two bright foci of H3K9me2 staining, corresponding to the unpaired regions of the X chromosomes (arrows). In some nuclei, these regions are not adjacent. Paired autosomes have weaker staining, as in wild-type. In contrast, the bright foci of H3K9me2 staining are not evident in ego-1;him-8 pachytene cells.

(C) ego-1/+;sDp3 animals have 1–2 bright foci of H3K9me2 staining in pachytene cells (arrows). Two foci result when partial pairing occurs between the left end of sDp3 and the left end of an intact chromosome III, leaving a portion of sDp3 and a portion of the other chromosome III unpaired. In contrast, ego-1;sDp3 pachytene cells lack the bright H3K9me2 foci on both sDp3 and the unpaired region of the intact chromosome III.

(D) Both dcr-1/+ and dcr-1 males have a bright focus of H3K9me2 staining in pachytene cells that corresponds to the tightly condensed X chromosome (arrows). Weaker staining is associated with autosomes.