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.

Author Summary

DNA within a cell's nucleus is packaged together with proteins into a higher order structure called chromatin. In its simplest form, chromatin consists of DNA and a set of proteins called histones, arranged so that the DNA strand is wrapped around histone protein clusters. This basic chromatin structure can be modified in various ways to regulate access to the genetic information encoded in the DNA. Such regulation is critical for cellular function and development of the organism. As cells form gametes, they undergo a specialized type of cell division called meiosis. During meiosis, chromatin is regulated in specific ways to ensure proper development of the embryo. During meiosis in the nematode C. elegans, the chromatin structure of the single male X chromosome depends on an RNA-directed RNA polymerase called EGO-1. Here, we identify three more regulators of meiotic chromatin, the proteins CSR-1, EKL-1, and DRH-3. Our data suggest that these proteins collaborate with EGO-1 to ensure that paired chromosomes (autosomes and hermaphrodite X chromosomes) are regulated correctly and in a manner distinct from the male X chromosome. Our findings suggest that these four proteins participate in a mechanism to ensure proper gene expression for gamete formation.