EGO-1, a putative RNA-dependent RNA polymerase, is required for heterochromatin assembly on unpaired dna during C. elegans meiosis

Abstract

During meiosis in C. elegans, unpaired chromosomes and chromosomal regions accumulate high levels of histone H3 lysine 9 dimethylation (H3K9me2), a modification associated with facultative heterochromatin assembly and the resulting transcriptional silencing. Meiotic silencing of unpaired DNA may be a widely conserved genome defense mechanism. The mechanisms of meiotic silencing remain unclear, although both transcriptional and posttranscriptional processes are implicated. Cellular RNA-dependent RNA polymerases (RdRPs) function in development and RNA-mediated silencing in many species and in heterochromatin assembly in S. pombe. There are four C. elegans RdRPs, including two with known germline functions. EGO-1 is required for fertility and robust germline RNAi. RRF-3 acts genetically to repress RNAi and is required for normal meiosis and spermatogenesis at elevated temperatures (S. L'Hernault, personal communication). Among C. elegans RdRPs, we find that only EGO-1 is required for H3K9me2 enrichment on unpaired chromosomal regions during meiosis. This H3K9me2 enrichment does not require Dicer or Drosha nuclease or any of several other proteins required for RNAi. ego-1 interacts genetically with him-17, another regulator of chromatin and meiosis, to promote germline development. We conclude that EGO-1 is an essential component of meiotic silencing in C. elegans.

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.

Figure 2. H3K9me2 Accumulation on Extrachromosomal, Multicopy Transgenes Is Defective in ego-1 but Not in dcr-1 Mutants

Each panel shows a portion of the meiotic germline from a strain carrying the repetitive extrachromosomal array, ex7291. (A) ex7291 is visible in DAPI-stained preparations of both ego-1/+ and ego-1 tissue (see insets; arrows indicate extrachromosomal array). A bright focus of H3K9me2 staining, corresponding to ex7291, is visible in ego-1/+ (arrows) but not ego-1 tissue. (B) A bright focus of H3K9me2 staining, corresponding to ex7291, is visible in both dcr-1/+ and dcr-1 tissue. Scale bar equals 20 μm; inset scale bar equals 5 μm.

Figure 3. ego-1 Interacts Genetically with him-17

Each panel shows one hermaphrodite gonad arm at 24 hr post-L4 stage. Asterisk indicates distal end of each arm. TZ, “transition zone” containing germ cells in early meiotic prophase (leptotene-zygotene stages). ego-1 and him-17 null alleles are shown (see Supplemental Experimental Procedures). Animals were raised at 20°C. (A) The wild-type germline includes distal mitotic cells, meiotic cells, and oocytes in diakinesis stage of meiosis. Sperm are present at the proximal end (although most are out of the plane of focus). (B) Organization of the him-17 germline is normal. (C) Overall organization of the ego-1 germline is normal, although some nuclei are enlarged, the leptotene-zygotene region is expanded, and oocytes are small. (D) The ego-1;him-17 germline is small and lacks oocytes. Sperm nuclei are irregular in size (inset).

Figure 4. Two Models for the Role of EGO-1 in Silencing Unpaired DNA during Meiosis

(A and B) Unpaired DNA (A) associates with and (B) is transcribed by a DNA-dependent RNA polymerase that is not active on paired DNA. HIM-17 may participate in this process. (C) EGO-1 binds to the RNA and recruits chromatin modifiers, e.g., histone methyltransferase (HMTase), to the region. RHA-1 may function in an RdRC-like complex with EGO-1 at this step. HMTase may directly bind to EGO-1 for its recruitment. Alternatively, EGO-1 generates dsRNA, which then triggers assembly of a RITS-like complex. RITS-like activity then recruits HMTase. (D) The end result of the EGO-1-RNA association is accumulation of H3K9me2 modifications preferentially on the unpaired DNA.