Carolyn Marks et al. (2002) West Coast Worm Meeting "Alae Mutant Defects Assessed by SEM and TEM"

Min Ren, Tylon Stephney, David H Hall, Carolyn Marks, Hui Feng, Charles S Rubin, Adam Hartley

[West Coast Worm Meeting, 2002]

We previously reported movement defects in adult C. elegans after RNAi (dsRNA feeding) with the gene PKC4, which encodes a novel Ser/Thr protein kinase (Ren et al., 2001). The PKC4 protein is expressed highly in the seam cells in late-stage embryos and again in late L4 stage, at the time of morphogenesis for seam-cell derived cuticular structures, the alae. It seemed likely that the movement defects, a sloppy zig-zag body motion of variable amplitude when placed on standard agar plates, were the result of absent or poorly formed alae. We have fixed adult animals for examination by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and compared these PKC4 RNAi animals to adult wild type. We will show SEM evidence for the normal extent of adult alae and several other hallmark features of the body surface, and TEM data for the corresponding seam cell cytology which underlies the alae. As predicted, the "knockout" animals often show alae defects or complete loss of alae locally, with a penetrance which agrees well with the prevalence of movement defects. The most common defect is a merger of the tripartite ala structure into a large smooth ridge of cuticle, or less often, a zone of smooth cuticle with no ridge at all. In severely affected PKC4 RNAi animals, some tissue defects can be seen in seam cells, which can wander away from the midline, or in the local hypodermis, which can show local swelling . Misassembled plaques of secreted material are sometimes seen in the cuticle in regions corresponding to the missing alae.

Booth LN et al. (2015) Mol Cell "Shockingly Early: Chromatin-Mediated Loss of the Heat Shock Response."

Booth LN, Brunet A

[Mol Cell, 2015]

In this issue of Molecular Cell, Labbadia and Morimoto (2015) show that there is a precipitous decline in stress resistance at the onset of reproduction in C.elegans and that this transition is regulated by changes in repressive chromatin marks.

Poulin, Gino et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Methylation and demethylation activities of a C. elegans MLL-like complex attenuate RAS signalling"

Fisher, Kate, Wilson, Jon, Poulin, Gino, Southall, Stacey

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The conserved Mixed Lineage Leukaemia (MLL) complex deposits activating methyl marks on histone tails through a methyltransferase (MT) activity. Here we provide in vivo evidence that in addition to methylation, the C. elegans MLL-like complex can remove specific methyl marks linked to repression of transcription. This supports the proposed model in which the MLL complex orchestrates both the deposition and the removal of methyl marks to activate transcription. We have uncovered the MLL-like complex in a large-scale RNAi screen designed to identify attenuators of RAS signalling during vulval development. We have also found that the histone acetyltransferase complex, NuA4/TIP60, cooperates with the C. elegans MLL-like complex in the attenuation of RAS signalling. Critically, we show that both complexes regulate a common novel target and attenuator of RAS signalling, AJM-1 (Apical Junction Molecule-1). Therefore, the C. elegans MLL-like complex cooperates with the NuA4/TIP60 complex to regulate the expression of a novel effector, AJM-1.

Burton NO et al. (2011) Proc Natl Acad Sci U S A "Nuclear RNAi maintains heritable gene silencing in Caenorhabditis elegans."

Kennedy S, Burkhart KB, Burton NO

[Proc Natl Acad Sci U S A, 2011]

RNA interference (RNAi) is heritable in Caenorhabditis elegans; the progeny of C. elegans exposed to dsRNA inherit the ability to silence genes that were targeted by RNAi in the previous generation. Here we investigate the mechanism of RNAi inheritance in C. elegans. We show that exposure of animals to dsRNA results in the heritable expression of siRNAs and the heritable deposition of histone 3 lysine 9 methylation (H3K9me) marks in progeny. siRNAs are detectable before the appearance of H3K9me marks, suggesting that chromatin marks are not directly inherited but, rather, reestablished in inheriting progeny. Interestingly, H3K9me marks appear more prominently in inheriting progeny than in animals directly exposed to dsRNA, suggesting that germ-line transmission of silencing signals may enhance the efficiency of siRNA-directed H3K9me. Finally, we show that the nuclear RNAi (Nrde) pathway maintains heritable RNAi silencing in C. elegans. The Argonaute (Ago) NRDE-3 associates with heritable siRNAs and, acting in conjunction with the nuclear RNAi factors NRDE-1, NRDE-2, and NRDE-4, promotes siRNA expression in inheriting progeny. These results demonstrate that siRNA expression is heritable in C. elegans and define an RNAi pathway that promotes the maintenance of RNAi silencing and siRNA expression in the progeny of animals exposed to dsRNA.

Fisher K et al. (2010) Dev Biol "Methylation and demethylation activities of a C. elegans MLL-like complex attenuate RAS signalling."

Wilson JR, Poulin GB, Southall SM, Fisher K

[Dev Biol, 2010]

The conserved Mixed Lineage Leukaemia (MLL) complex deposits activating methyl marks on histone tails through a methyltransferase (MT) activity. Here we provide in vivo evidence that in addition to methylation, the C. elegans MLL-like complex can remove specific methyl marks linked to repression of transcription. This supports the proposed model in which the MLL complex orchestrates both the deposition and the removal of methyl marks to activate transcription. We have uncovered the MLL-like complex in a large-scale RNAi screen designed to identify attenuators of RAS signalling during vulval development. We have also found that the histone acetyltransferase complex, NuA4/TIP60, cooperates with the C. elegans MLL-like complex in the attenuation of RAS signalling. Critically, we show that both complexes regulate a common novel target and attenuator of RAS signalling, AJM-1 (Apical Junction Molecule-1). Therefore, the C. elegans MLL-like complex cooperates with the NuA4/TIP60 complex to regulate the expression of a novel effector, AJM-1.

Kelly WG (2014) Dev Cell "Multigenerational chromatin marks: no enzymes need apply."

Kelly WG

[Dev Cell, 2014]

Epigenetic memory stably maintains and transmits information during genome replication. Recently in Science, Gaydos etal. (2014) show that repressive chromatin marks exhibit transgenerational stability in the absence of chromatin-modifying enzymes in Caenorhabditis elegans, in contrast to work in flies suggesting that such proteins mediate stable inheritance of epigenetic modifications.

Allo M et al. (2010) Curr Biol "Gene silencing: small RNAs control RNA polymerase II elongation."

Allo M, Kornblihtt AR

[Curr Biol, 2010]

Short interfering RNAs trigger histone silencing marks and stalling of RNA polymerase II at their genomic target sites through a mechanism termed transcriptional gene silencing (TGS). The Argonaute protein NRDE-3, along with NRDE-2, are needed for TGS in C. elegans. TGS also inhibits elongation and controls alternative splicing in mammalian cells.

Guo, Yiqing et al. (2013) International Worm Meeting "Differential expression of germline genes in the presence/absence of H3K9me2."

Maine, Eleanor, Guo, Yiqing

[International Worm Meeting, 2013]

In C. elegans, indirect immunofluorescence experiments indicate a high level of histone H3 lysine 9 dimethylation (H3K9me2) on any unpaired/unsynapsed chromosomes during first meiotic prophase, particularly in pachytene stage nuclei (Kelly et al 2002; Bean et al 2004). In contrast, H3K9me2 marks are relatively low on paired chromosomes at this time. H3K9me2 marks are typically associated with transcriptional repression. Thus, the elevated level of H3K9me2 on unpaired chromosomes may play a role in meiotic silencing of unpaired chromosomes (MSUC). Germline H3K9me2 marks require activity of MET-2 (Bessler et al 2010), a SET domain protein that is the only C. elegans ortholog of human SETDB1 H3K9 methyltransferase (Schultz et al 2002). To begin to investigate the importance of H3K9me2 marks on unpaired chromosomes, we performed RNA-seq on mRNA isolated from him-8 vs met-2;him-8 adult hermaphrodite gonads. Our data identify genes that are expressed in the gonads and that differentially expressed in met-2(0) and met 2(+). In most cases, the differentially expressed transcripts are elevated in met-2(0), consistent with a role for H3K9me2 in limiting transcription. Although elevated transcripts are not preferentially X-linked, we observe a greater average fold-change for X-linked transcripts than for autosomal transcripts. To evaluate whether the unpaired status of the X contributes to this pattern, we are evaluating additional genotypes. As a complementary approach, we are also investigating the distribution of H3K9me2 on paired vs unpaired chromosomes using a ChIP-seq approach.

Lan KY et al. (2021) Comput Struct Biotechnol J "Epigenomic signatures on paralogous genes reveal underappreciated universality of active histone codes adopted across animals."

Liao BY, Lan KY

[Comput Struct Biotechnol J, 2021]

The results of conventional gene-based analyses which combine epigenome and transcriptome data, including those conducted by the ENCODE/modENCODE projects, suggest various histone modifications performing regulatory functions in controlling mRNA expression (referred to as a histone code) in several model animals. While some histone codes were found to be universally adopted across organisms, "species-specific" histone codes have also been defined. We found that the characterization of these histone codes was confounded by factors (e.g. gene essentiality, expression breadth) that are independent of, but correlated with, gene expression levels. Hence, we attempted to decode histone marks in mouse (<i>Mus musculus</i>), fly (<i>Drosophila melanogaster</i>), and worm (<i>Caenorhabditis elegans</i>) genomes by examining ratios of RNA sequencing (and chromatin immunoprecipitation sequencing) intensities between paralog genes to remove confounding effects that would otherwise be present in a gene-based approach. With this paralog-based approach, associations between four histone modifications (H3K4me3, H3K27ac, H3K9ac, and H3K36me3) and gene expression are substantially revised. For example, we demonstrate that H3K27ac and H3K9ac represent universal active marks in promoters, rather than worm-specific marks as previously reported. Second, acting regions of the studied active marks that are common across species (and across a wide range of tissues at different developmental stages) were found to extend beyond the previously defined regions. Thus, it appears that the active histone codes analyzed have a universality that has previously been underappreciated. Our results suggested that these universal codes, including those previously considered species-specific, could have an ancient origin, and are important in regulating animal gene expression abundance.

Guerin TM et al. (2014) Epigenetics "Transgenerational functions of small RNA pathways in controlling gene expression in C. elegans."

Robert VJ, Palladino F, Guerin TM

[Epigenetics, 2014]

RNA silencing processes use exogenous or endogenous RNA molecules to specifically and robustly regulate gene expression. In C. elegans, initial mechanistic descriptions of the different silencing processes focused on posttranscriptional regulation. In this review, we discuss recent work showing that, in this model organism, RNA silencing also controls the transcription of target genes by inducing heterochromatin formation. Specifically, it has been shown that ribonucleoprotein complexes containing small RNAs, either processed from exogenous dsRNA or synthesized from the genome itself, and proteins of the Argonaute family, mediate the deposition of repressive histone marks at the targeted loci. Interestingly, the accumulation of repressive marks is required for the inheritance of the silencing effect and the establishment of an epigenetic memory that discriminates self- from non-self-RNAs.