Repressive Chromatin in Caenorhabditis elegans: Establishment, Composition, and Function

Abstract

Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals.

Keywords: C. elegans; H3K27me; H3K9me; WormBook; chromatin; heterochromatin; histone methylation.

Figure 1

Histone modifications associated with heterochromatin and genome domains. Top: pattern of histone modifications associated with heterochromatin (H3K9me2, H3K9me3, and H3K27me3), H3K36me3, and LEM-2 domains (indicating nuclear lamina association) in embryos across C. elegans chromosome II. The majority of H3K9me marks and LEM-2 domains are found on the chromosome arms. H3K27me3 levels are higher on arm regions compared to the center. H3K36me3 shows a more uniform pattern, with a slight enrichment in the central region. Bottom left: 450-kb segment of a central region showing low H3K9 methylation, and anticorrelated domains marked by H3K27me3 and H3K36me3. H3K36me3 is found in active chromatin domains (orange) and H3K27me3 in regulated chromatin domains (black), which are separated by border regions (gray). Bottom right: 450-kb segment of an arm region. The distributions of H3K9me3 and H3K27me3 are largely similar to each other, but differ from H3K9me2. Patterns of H3K36me3 and H3K27me3 and chromatin domains are similar to those in central regions.

Figure 2

Histone H3K9 methylation triggers peripheral localization of chromatin independently of HPL-1/-2 or LIN-61 binding. In C. elegans early embryos, CEC-4 recognizes and binds H3K9 me1, me2, or me3 to mediate the anchoring of appropriately modified nucleosomes to the nuclear periphery, without necessarily repressing transcription (Gonzalez-Sandoval et al. 2015). The H3K9me ligands HPL-2 and LIN-61 mediate transcriptional repression by binding H3K9 methylation, but do not anchor chromatin. HPL-1 is associated with repressed chromatin, but its role in repression remains unclear. SET-25 colocalizes with heterochromatic transgene arrays bearing H3K9me3, and its activity, along with HPL-2 and LIN-61, leads to repression. MET-2 and/or SET-25 deposit H3K9me1 and me2, while only SET-25 deposits H3K9me3 in somatic cells (Towbin et al. 2012). Alternative anchors may be present in differentiated cells, although their identity is unknown.

Figure 3

Larvae lacking MET-2 and SET-25 show an accumulation of RNA:DNA hybrids or R-loops at transcribed repeat elements. (A) Genome-wide distribution of R-loops determined by RNA:DNA immunoprecipitation (DRIP) with antibody S9.6, followed by deep sequencing of recovered DNA [see Zeller et al. (2016) for details]. Heat map of an S9.6 DRIP sequencing (DRIP-seq) experiment showing mean log2 enrichment over the corresponding RNaseH-treated controls. Loci are segregated based on the type of repeat element (vertical legend) and whether or not the sequences carry H3K9 methylation in wild-type cells (by chromatin immunoprecipitation analysis, labeled H3K9me+), or whether they were derepressed in set-25 met-2 vs. wild-type embryos by RNA sequencing (horizontal legend). The lower panel is a DRIP-seq example showing the R-loop signal over a repeat element cluster. The immunoprecipitation signal is normalized to the input and the RNaseH control values were subtracted. (B) Transcribed repeat elements in H3K9me-deficient strains can exacerbate replication stress, provoking insertions and deletions in repetitive parts of the genome. This model suggests that the loss of H3K9me leads to R-loops, which allows the formation of secondary DNA structures that engender fork arrest, slippage, and breakage as forks deal with replication stress (Aguilera and Garcia-Muse 2012). These may perturb genome integrity, especially at heterochromatic repeats. Modified from Zeller et al. (2016). Chrl, Chromosome 1; RNAPII, RNA polymerase II.

Figure 4

Functional antagonism between MES-4 and PRC2. The genome is organized into domains of genes expressed only in the germ line, or both in the germ line and broadly across cell types, and domains of genes with somatic cell and regulated expression. MES-4 marks genes transcribed in the germ line with H3K36me2/3. This includes germ line-specific genes (red) and broadly expressed genes (orange). A PRC2-like complex composed of MES-2, MES-3, and MES-6 marks somatic genes (gray) with H3K27me3. MES-4 inhibits PRC2: in mes-4(RNAi) embryos (derived from RNA interference-treated mothers), H3K27me3 marking spreads into regions previously occupied by H3K36me3.

Figure 5

Heterochromatin proteins and small RNA pathways repress transcription of repetitive elements and genes. Repressed chromatin is marked by methylation of H3K9me2 and/or H3K9me3. The nuclear RNAi (Nrde) pathway uses small RNAs bound by argonaute proteins HRDE-1 (germ line) or NRDE-3 (soma) to target NRDE proteins to chromatin, leading to inhibition of transcription elongation and H3K9me3 marking, dependent on SET-25. Some H3K9me3 induced by exogenous application of RNAi is dependent on SET-32. Loss of NRDE function leads to derepression of repetitive elements (enriched for those derived from retrotransposons) and genes. The piRNA pathway, initiated in the germ line cytoplasm, targets genes and repetitive elements for repression by transcriptional and post-transcriptional mechanisms. The piRNA pathway engages the Nrde pathway for transcriptional silencing through the generation of small RNAs that bind HRDE-1. A set of heterochromatin factors are found together at many genomic locations and are expressed in the germ line and soma. They repress common elements by a mechanism that is not yet understood. Repetitive elements that require the heterochromatin proteins for repression (enriched for DNA transposons) largely differ from those requiring the Nrde pathway. This apparent difference appears to be at least partially due to functional redundancy between the Nrde pathway and heterochromatin factors, the nature of which is not yet known. piRNA, piwi-interacting RNA; RNAi, RNA interference; RNAPII, RNA polymerase II.