Devanapally, S. et al. (2017) International Worm Meeting "Transgenerational silencing in C. elegans can be triggered by mating."

Jose, A.M., Devanapally, S., Allgood, S.

[International Worm Meeting, 2017]

Silencing of foreign DNA sequences is a significant obstacle to genome engineering and has been interpreted to be the result of the organism mounting a non-self response through studies in the worm C. elegans. This hypothesis does not explain how some sequences apparently defy recognition as foreign by the organism and remain stably expressed for many generations. Here, we report a phenomenon where the expression of a foreign sequence in C. elegans can be heritably silenced upon mating with wild-type animals. Sibling progeny from a mating differ in their ability to initiate and transmit silencing across generations, suggesting that the phenotype of an animal can depend on the ancestral progeny selected from a mating. Dissection of this mating-induced transgenerational silencing revealed two different DNA-independent signals that control gene expression: a protective signal transmitted through oocytes that prevents initiation of silencing, and a silencing signal transmitted through both oocytes and sperm that can act on homologous sequences. Transgenerational silencing relies on at least two classes of small RNAs because it requires the Argonaute PRG-1 for initiation but the Argonaute HRDE-1 for maintenance. Silencing is actively maintained in each generation because loss of HRDE-1 can revive gene expression even after 150 generations. Our results signal caution in the interpretation of observations that rely on genetic crosses and reveal powerful epigenetic mechanisms that can dictate phenotype in response to ancestral mating.

Devanapally S et al. (2021) Nat Commun "Mating can initiate stable RNA silencing that overcomes epigenetic recovery."

Raman P, Diop M, Ettefa F, Lin Y, Chey M, Allgood S, Jose AM, Devanapally S, Cho YE

[Nat Commun, 2021]

Stable epigenetic changes appear uncommon, suggesting that changes typically dissipate or are repaired. Changes that stably alter gene expression across generations presumably require particular conditions that are currently unknown. Here we report that a minimal combination of cis-regulatory sequences can support permanent RNA silencing of a single-copy transgene and its derivatives in C. elegans simply upon mating. Mating disrupts competing RNA-based mechanisms to initiate silencing that can last for >300 generations. This stable silencing requires components of the small RNA pathway and can silence homologous sequences in trans. While animals do not recover from mating-induced silencing, they often recover from and become resistant to trans silencing. Recovery is also observed in most cases when double-stranded RNA is used to silence the same coding sequence in different regulatory contexts that drive germline expression. Therefore, we propose that regulatory features can evolve to oppose permanent and potentially maladaptive responses to transient change.

Devanapally, S et al. (2019) International Worm Meeting "Epigenetic recovery from transgenerational RNA silencing in the C. elegans germline."

Lin, Y, Chey, M, Ettefa, F, Cho, Y, Jose, A, Devanapally, S, Diop, M, Allgood, S, Raman, P

[International Worm Meeting, 2019]

Changes in gene expression that last for multiple generations without changes in gene sequence have been reported in many plants and animals. Cases of such transgenerational epigenetic inheritance (TEI) have been proposed to support the ancestral origins of some diseases and to drive evolutionary novelty. Here, we report that stably expressed sequences in C. elegans have features that provide a barrier against TEI. By using double-stranded RNA (dsRNA) targeting the same sequence in different genes, we show that genes typically recover from silencing within the germline in a few generations. A rare recombinant gene that recovers poorly but is stably expressed in the absence of induced changes has enabled us to delineate multiple mechanisms that can perpetuate silencing. Parental exposure to dsRNA targeting one gene of a two-gene operon reveals two distinct phases of the resulting TEI: only the matching gene is silenced in the first two generations, but both are silenced in later generations. However, silencing of both genes can be initiated within one generation by mating, which perturbs intergenerational RNA-based mechanisms such that silencing dominates for more than 250 generations. This stable RNA silencing can also reduce the expression of homologous sequences in different genes within the germline, but the homologous genes recover expression after a few generations. These results suggest that most stably expressed sequences are subject to feedback control that opposes TEI initiated by multiple mechanisms within the germline. We speculate that similar homeostatic mechanisms that allow recovery from epigenetic changes underlie the observed preservation of form and function in successive generations of living systems.

Devanapally S et al. (2015) Proc Natl Acad Sci U S A "Double-stranded RNA made in C. elegans neurons can enter the germline and cause transgenerational gene silencing."

Ravikumar S, Devanapally S, Jose AM

[Proc Natl Acad Sci U S A, 2015]

An animal that can transfer gene-regulatory information from somatic cells to germ cells may be able to communicate changes in the soma from one generation to the next. In the worm Caenorhabditis elegans, expression of double-stranded RNA (dsRNA) in neurons can result in the export of dsRNA-derived mobile RNAs to other distant cells. Here, we show that neuronal mobile RNAs can cause transgenerational silencing of a gene of matching sequence in germ cells. Consistent with neuronal mobile RNAs being forms of dsRNA, silencing of target genes that are expressed either in somatic cells or in the germline requires the dsRNA-selective importer SID-1. In contrast to silencing in somatic cells, which requires dsRNA expression in each generation, silencing in the germline is heritable after a single generation of exposure to neuronal mobile RNAs. Although initiation of inherited silencing within the germline requires SID-1, a primary Argonaute RDE-1, a secondary Argonaute HRDE-1, and an RNase D homolog MUT-7, maintenance of inherited silencing is independent of SID-1 and RDE-1, but requires HRDE-1 and MUT-7. Inherited silencing can persist for >25 generations in the absence of the ancestral source of neuronal dsRNA. Therefore, our results suggest that sequence-specific regulatory information in the form of dsRNA can be transferred from neurons to the germline to cause transgenerational silencing.

Ravikumar S et al. (2019) Nucleic Acids Res "Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference."

Jose AM, Ravikumar S, Devanapally S

[Nucleic Acids Res, 2019]

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.

Ravikumar, S et al. (2017) International Worm Meeting "Differential requirements for an RNA-dependent RNA polymerase during RNA interference within C. elegans somatic cells."

Devanapally, S, Jose, AM, Ravikumar, S

[International Worm Meeting, 2017]

Double-stranded RNA (dsRNA) from diverse sources can trigger RNA interference in C. elegans. These include dsRNA transcribed within bacteria that worms ingest, in vitro transcribed dsRNA introduced into worms by ingestion or injection, and in vivo transcribed dsRNA made in the same cell as the target gene to be silenced or in a distant cell that exports dsRNA to the cell with the target gene. It is generally assumed that all these forms of dsRNA delivery engage the same mechanism to cause gene silencing. Intriguingly, we found that the need for an RNA-dependent RNA polymerase (RdRP) for silencing the same target gene can differ based on the mode of delivery of dsRNA. While ingested dsRNA expressed in bacteria and intracellularly transcribed dsRNA both required the RdRP RRF-1 to silence a multi-copy transgene expressing GFP in intestinal cells, gfp-dsRNA from neurons did not require RRF-1 for silencing the same target or a single-copy transgene expressing GFP in all cells. However, not all neuronally expressed dsRNA caused RRF-1-independent silencing in a distant tissue. Silencing of the endogenous muscle gene unc-22 by unc-22-dsRNA from neurons was entirely RRF-1 dependent. These differential requirements could reflect differences in amounts of dsRNA being delivered, molecular features of dsRNA being delivered, molecular features of the target gene, or tissue-specific variation in silencing mechanisms. To explicitly test each possibility, we will generate strains with different dsRNA sources and target gene contexts using CRISPR-based genome editing. For example, we have created a chimeric target composed of both unc-22 and gfp to test if having both targets as a single transcript eliminates the differences in RRF-1 requirement for silencing by unc-22-dsRNA or gfp-dsRNA from neurons. Results of these experiments and subsequent analyses will be presented at the meeting.

Ravikumar, S. et al. (2015) International Worm Meeting "Mobile RNA and intracellularly transcribed RNA silence genes using distinct mechanisms in C.elegans."

Jose, A.M., Ravikumar, S., Devanapally, S.

[International Worm Meeting, 2015]

The export of RNA from a cell can result in sequence-specific regulation in distant cells. In the worm C. elegans, expression of double-stranded RNA (dsRNA) in neurons results in the export of forms of dsRNA called mobile RNA to enable silencing of a gene of matching sequence in another cell through RNA interference (RNAi). When dsRNA is present within a somatic cell, it is processed into primary small interfering RNA that are used by the Argonaute RDE-1 to recruit the RNA-dependent RNA polymerse (RdRP) RRF-1 to the target mRNA for secondary small interfering RNA production and subsequent silencing. However, it is unknown whether silencing in response to mobile RNA and intracellularly transcribed RNA relies on the same mechanisms. To understand silencing of a single gene within cells that encounter both mobile RNA and intracellularly transcribed dsRNA, we generated a strain where neuronal mobile RNA against a transgene coding for green fluorescent protein (gfp) and intracellularly transcribed dsRNA from the repetitive gfp transgene together robustly silence gfp expression in gut cells in an enhanced RNAi (eri-1(-)) background. In a forward genetic screen for silencing by eri-1 loss or by neuronal dsRNA (send) defective mutants using this strain, we obtained one rde-1 mutant and three rrf-1 mutants. Analysis of these mutants and null alleles of rde-1 and rrf-1 revealed that while both sources of dsRNA require the Argonaute RDE-1 for silencing, only silencing by neuronal mobile RNA was entirely independent of the RdRP RRF-1. Intriguingly, this RRF-1-independent silencing of a target gene by neuronal mobile RNA was context-dependent. Silencing of the endogenous muscle gene unc-22 by neuronal mobile RNA against unc-22 was RRF-1-dependent but silencing of a ubiquitously-expressed single-copy gfp transgene by neuronal mobile RNA against gfp was RRF-1-independent. Our results suggest that mobile RNA and intracellular RNA can differ in their use of RRF-1 for silencing the same gene and future work will focus on understanding the basis of this differential requirement for RRF-1.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Termine D, Becuwe M, Hofbauer HF, Barrasa MI, Pincus D, Imberdis T, Selkoe D, Freyzon Y, Srinivasan S, Soldner F, Nuber S, Sandoe J, Haque A, Welte MA, Clish CB, Terry-Kantor E, Jaenisch R, Kohlwein SD, Fanning S, Dettmer U, Walther TC, Kim TE, Farese RV, Landgraf D, Baru V, Noble T, Lou Y, Lindquist S, Newby G, Ho GPH, Ramalingam N

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Coenye T, Van Nieuwerburgh F, Deforce D, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.