Regulation of heterochromatin assembly on unpaired chromosomes during Caenorhabditis elegans meiosis by components of a small RNA-mediated pathway

Abstract

Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome) become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2). This enrichment requires activity of the cellular RNA-directed RNA polymerase, EGO-1. Here we use genetic mutation, RNA interference, immunofluorescence microscopy, fluorescence in situ hybridization, and molecular cloning methods to identify and analyze three additional regulators of meiotic H3K9me2 distribution: CSR-1 (a Piwi/PAZ/Argonaute protein), EKL-1 (a Tudor domain protein), and DRH-3 (a DEAH/D-box helicase). In csr-1, ekl-1, and drh-3 mutant males, we observed a reduction in H3K9me2 accumulation on the unpaired X chromosome and an increase in H3K9me2 accumulation on paired autosomes relative to controls. We observed a similar shift in H3K9me2 pattern in hermaphrodites that carry unpaired chromosomes. Based on several assays, we conclude that ectopic H3K9me2 accumulates on paired and synapsed chromosomes in these mutants. We propose alternative models for how a small RNA-mediated pathway may regulate H3K9me2 accumulation during meiosis. We also describe the germline phenotypes of csr-1, ekl-1, and drh-3 mutants. Our genetic data suggest that these factors, together with EGO-1, participate in a regulatory network to promote diverse aspects of development.

Figure 1. drh-3 and ekl-1 mutations isolated as genetic enhancers of glp-1.

The locations of ego alleles within (A) drh-3 and (B) ekl-1 are shown. ego alleles were isolated in screens for genetic enhancers of glp-1 sterility. Annotated open reading frame designation (e.g., D2005.5) and nucleotide position on LGI (e.g., I:7820836) are indicated. Boxes represent exons and lines represent introns. Nucleotide and amino acid changes are indicated. The position of the drh-3(tm1217) deletion is indicated for reference. 3′ UTRs are shaded yellow; 5′ UTRs are not represented.

Figure 2. Abnormal H3K9me2 accumulation in csr-1, ekl-1, and drh-3 mutants.

Each panel shows germline nuclei co-labeled with DAPI to visualize DNA and polyclonal anti-H3K9me2 antibody. Tissue was dissected and fixed at 24 hr post-L4 stage. The distal-to-proximal axis is oriented left to right in each image. All images were taken at the same exposure. (A and B) Features of the wild type (N2) and csr-1(tm892) male germ lines are shown. Distal ends (asterisk), mitotic zones (MZ), transition zones (TZ), pachytene zones (PZ), and primary spermatocytes are indicated. In both germ lines, H3K9me2 is first detected at early pachytene stage. (A') In the N2 germ line, a single strong focus of H3K9me2 labeling corresponds to the single X chromosome (as described in [4]); other chromosomes accumulate a low level of H3K9me2 (as described in [9]). (B) In the csr-1(tm892) germ line, H3K9me2 labeling is more broadly distributed than in wildtype; a single strong focus of labeling is often absent. (B') An example of a large, morphologically abnormal nucleus is circled. See Results. (B'″) As in wildtype, H3K9me2 levels decrease as germ cells become primary spermatocytes. Circled nucleus has the “elevated” H3K9me2 pattern referred to in Table 2 and described in Results. (C–I) Each panel shows H3K9me2 distribution in pachytene nuclei at a position corresponding to the region shown in (B″-B'″). (C) The X chromosome fails to accumulate a high level of H3K9me2 in ego-1(om84) meiotic nuclei; a basal level of H3K9me2 is broadly distributed over all chromosomes. (D,E) H3K9me2 distribution in drh-3(tm1217) and ekl-1(om56) single mutants resembles that observed in csr-1(tm892) animals (see (B″)). (F–I) H3K9me2 distribution in ego-1;csr-1, ego-1 drh-3, ekl-1 ego-1, and ekl-1 drh-3 double mutants resembles that observed in csr-1, ekl-1, and drh-3 single mutants. Images were captured on a Zeiss Axioscope.

Figure 3. Relative distribution of H3K4me2 and H3K9me2 marks during XO meiosis.

Each panel shows meiotic nuclei co-labeled with DAPI to visualize DNA, polyclonal antibody against H3K4me2, and monoclonal antibody against H3K9me2. In wildtype germ cells, the X chromosome (arrow) lacks H3K4me2 (*) and becomes highly enriched for H3K9me2 (arrow). In contrast, autosomes become highly enriched for H3K4me2 and accumulate a very low level of H3K9me2. In csr-1, ekl-1, and drh-3 nuclei, one chromosomal region lacks H3K4me2 (*) and contains a variable level of H3K9me2 (arrow); this is presumably the X chromosome. In some cases, the X chromosome lacks detectable H3K9me2 altogether (*). Other chromosomes contain substantial H3K4me2 and a variable level of H3K9me2 (arrowheads). Circled nuclei are examples of the “elevated” H3K9me2 pattern referred to in Table 2; most other nuclei have the “dispersed” H3K9me2 pattern referred to in Table 2. See Results. Images were captured on a Zeiss Axioscope at the same exposure and processed in a similar manner.

Figure 4. Reduced H3K9me2 levels in csr-1, ekl-1, and drh-3 hermaphrodites carrying unpaired X chromosomes or a chromosomal duplication.

Each panel shows hermaphrodite germline nuclei co-labeled with DAPI to visualize DNA and with polyclonal anti-H3K9me2 antibody. All images were taken at the same exposure. Unpaired chromatin was introduced into hermaphrodite germ cells using (A) a him-8 mutation to prevent X chromosome pairing or (B) the free duplication, sDp3. (A) In him-8 controls, H3K9me2 foci are visible (arrowheads). In drh-3;him-8 and ekl-1;him-8 mutants, unpaired X chromosomes fail to become enriched for H3K9me2 (arrows). (B) In control nuclei, H3K9me2 is elevated on sDp3 (fat arrows); occasional nuclei have two foci that are interpreted to reflect partial pairing of sDp3 with an intact LGIII, resulting in H3K9me2 enrichment on the unpaired portions of sDp3 and LGIII. In sDp3;csr-1 and ekl-1;sDp3 nuclei, the H3K9me2 labeling is no longer concentrated as a single strong focus, but instead is found on multiple chromosomes (arrows) and in some cases is reduced on sDp3 relative to wild type. Images were captured on a Zeiss Axioscope.

Figure 5. Examples of gametogenesis defects associated with loss of csr-1, drh-3, and ekl-1 function.

Each panel shows a portion of a dissected gonad arm stained with DAPI to visualize DNA. Known or putative null alleles are used in all cases. (A) Wildtype (N2) hermaphrodite with oocyte nuclei (open arrowheads) and sperm (Sp, line) are indicated. Note that sperm are essentially uniform in size. Oocyte nuclei at diakinesis have six bivalent chromosomes. (B) ego-1 sperm are essentially uniform in size. (C) ekl-1drh-3, (E) ego-1drh-3, and (H) ekl-1 germ lines contain small, crowded oocytes with univalent chromosomes (closed arrowheads). (C) ekl-1drh-3, (D) drh-3, (E) ego-1drh-3, and (F') (some) ekl-1 ego-1 germ lines contain variably sized sperm (arrows). (F) Some ekl-1 ego-1 germ lines contain sperm of essentially uniform size. (E and G) Morphologically abnormal nuclei are visible amidst diakinesis (oocyte) nuclei in ego-1drh-3 and csr-1 germ lines (fat arrows). In addition, csr-1 meiotic nuclei are often clumped together, leaving gaps without nuclei (bracket).

Figure 6. Distribution of H3K9me2 relative to 5S rDNA FISH signal.

Panels show representative pachytene nuclei from a drh-3 XO germ line co-labeled to detect 5S rDNA (by FISH) and H3K9me2. (A,C) The single FISH signal is adjacent to a region with high H3K9me2 label. (B) Two FISH signals are detected, one of which is well-separated from regions of high H3K9me2 label. Images were captured on a Leica DRMXA microscope.

Figure 7. SYP-1 vs H3K9me2 distribution in csr-1, ekl-1, and drh-3 mutants.

Each panel shows pachytene nuclei in an adult germ line co-labeled with DAPI to visualize DNA, anti-SYP-1 to visualize the synaptonemal complex, and anti-H3K9me2. As indicated in Figure S2, Figure S3, and Figure S4, SYP-1 is associated with all chromosomes except the partnerless male X (arrows). In wildtype male germ cells, the strong focus of H3K9me2 staining corresponds to the X chromosome (arrows). In mutants, H3K9me2 foci can be found associated with SYP-1 (arrowheads), indicating H3K9me2 enrichment on synapsed chromosomes. Images were captured on a Zeiss Axioscope.