Evolution beyond DNA: epigenetic drivers for evolutionary change?

Epigenetics has been a buzzword in science for many years, but 20 years ago, the idea that epigenetic gene regulation could be a driver in evolutionary processes was only a mischievous glint in a Lamarckian eye. Now, however, theoretical and experimental studies have illuminated the possible roles that epigenetic information could play in evolution and we are on the cusp of testing this concept in ecologically relevant settings

The curious case of the disappearing piRNAs

Small non-coding RNAs are key regulators of gene expression across eukaryotes. Piwi-interacting small RNAs (piRNAs) are a specific type of small non-coding RNAs, conserved across animals, which are best known as regulators of genome stability through their ability to target transposable elements for silencing. Despite the near ubiquitous presence of piRNAs in animal lineages, there are some examples where the piRNA pathway has been lost completely, most dramatically in nematodes where loss has occurred in at least four independent lineages. In this perspective I will provide an evaluation of the presence of piRNAs across animals, explaining how it is known that piRNAs are missing from certain organisms. I will then consider possible explanations for why the piRNA pathway might have been lost and evaluate the evidence in favor of each possible mechanism. While it is still impossible to provide definitive answers, these theories will prompt further investigations into why such a highly conserved pathway can nevertheless become dispensable in certain lineages. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution

Histone methyltransferase activity affects metabolism in human cells independently of transcriptional regulation

The N-terminal tails of eukaryotic histones are frequently posttranslationally modified. The role of these modifications in transcriptional regulation is well-documented. However, the extent to which the enzymatic processes of histone posttranslational modification might affect metabolic regulation is less clear. Here, we investigated how histone methylation might affect metabolism using metabolomics, proteomics, and RNA-seq data from cancer cell lines, primary tumour samples and healthy tissue samples. In cancer, the expression of histone methyltransferases (HMTs) was inversely correlated to the activity of NNMT, an enzyme previously characterised as a methyl sink that disposes of excess methyl groups carried by the universal methyl donor S-adenosyl methionine (SAM or AdoMet). In healthy tissues, histone methylation was inversely correlated to the levels of an alternative methyl sink, PEMT. These associations affected the levels of multiple histone marks on chromatin genome-wide but had no detectable impact on transcriptional regulation. We show that HMTs with a variety of different associations to transcription are co-regulated by the Retinoblastoma (Rb) tumour suppressor in human cells. Rb-mutant cancers show increased total HMT activity and down-regulation of NNMT. Together, our results suggest that the total activity of HMTs affects SAM metabolism, independent of transcriptional regulation

Malignancy and NF-κB signalling strengthen coordination between expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes

Background Mitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. The mammalian mitochondrial genome encodes for 13 mitochondrial proteins, and the remaining mitochondrial proteins are encoded by the nuclear genome. Little is known about how coordination between the expression of the two sets of genes is achieved. Results Correlation analysis of RNA-seq expression data from large publicly available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalisation techniques based on total read counts, such as FPKM or TPM, produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, with important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalisation (MRN) or trimmed mean of M values (TMM) methods. Using these normalisations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database. Conclusions We show that a subset of healthy tissues with high expression of NF-κB show significant coordination, suggesting a role for NF-κB in ensuring balanced expression between mitochondrial and nuclear genes. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer

Cisplatin exposure alters tRNA-derived small RNAs but does not affect epimutations in C. elegans

Background The individual lifestyle and environment of an organism can influence its phenotype and potentially the phenotype of its offspring. The different genetic and non-genetic components of the inheritance system and their mutual interactions are key mechanisms to generate inherited phenotypic changes. Epigenetic changes can be transmitted between generations independently from changes in DNA sequence. In Caenorhabditis elegans, epigenetic differences, i.e. epimutations, mediated by small non-coding RNAs, particularly 22G-RNAs, as well as chromatin have been identified, and their average persistence is three to five generations. In addition, previous research showed that some epimutations had a longer duration and concerned genes that were enriched for multiple components of xenobiotic response pathways. These results raise the possibility that environmental stresses might change the rate at which epimutations occur, with potential significance for adaptation. Results In this work, we explore this question by propagating C. elegans lines either in control conditions or in moderate or high doses of cisplatin, which introduces genotoxic stress by damaging DNA. Our results show that cisplatin has a limited effect on global small non-coding RNA epimutations and epimutations in gene expression levels. However, cisplatin exposure leads to increased fluctuations in the levels of small non-coding RNAs derived from tRNA cleavage. We show that changes in tRNA-derived small RNAs may be associated with gene expression changes. Conclusions Our work shows that epimutations are not substantially altered by cisplatin exposure but identifies transient changes in tRNA-derived small RNAs as a potential source of variation induced by genotoxic stress

E. coli OxyS non-coding RNA does not trigger RNAi in C. elegans.

The discovery of RNA interference (RNAi) in C. elegans has had a major impact on scientific research, led to the rapid development of RNAi tools and has inspired RNA-based therapeutics. Astonishingly, nematodes, planaria and many insects take up double-stranded RNA (dsRNA) from their environment to elicit RNAi; the biological function of this mechanism is unclear. Recently, the E. coli OxyS non-coding RNA was shown to regulate gene expression in C. elegans when E. coli is offered as food. This was surprising given that C. elegans is unlikely to encounter E. coli in nature. To directly test the hypothesis that the E. coli OxyS non-coding RNA triggers the C. elegans RNAi pathway, we sequenced small RNAs from C. elegans after feeding with bacteria. We clearly demonstrate that the OxyS non-coding RNA does not trigger an RNAi response in C. elegans. We conclude that the biology of environmental RNAi remains to be discovered.PS is funded by a research fellowship from the Gonville and Caius College, University of Cambridge. AA and EAM are supported by a Cancer Research UK programme grant to EAM.This is the final published version. It first appeared at http://www.nature.com/srep/2015/150411/srep09597/full/srep09597.html

Identification of proteins that bind extracellular microRNAs secreted by the parasitic nematode Trichinella spiralis

Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes, and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently, we characterised the parasitic nematode Trichinella spiralis as a model to study miRNA secretion. T. spiralis muscle-stage larvae (MSL) secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds to miRNA without exhibiting sequence specificity. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely

Gene body methylation evolves during the sustained loss of parental care in the burying beetle

Epigenetic modifications, such as 5-methylcytosine (5mC), can sometimes be transmitted between generations, provoking speculation that epigenetic changes could play a role in adaptation and evolution. Here, we use experimental evolution to investigate how 5mC levels evolve in populations of biparental insect (Nicrophorus vespilloides) derived from a wild source population and maintained independently under different regimes of parental care in the lab. We show that 5mC levels in the transcribed regions of genes (gene bodies) diverge between populations that have been exposed to different levels of care for 30 generations. These changes in 5mC do not reflect changes in the levels of gene expression. However, the accumulation of 5mC within genes between populations is associated with reduced variability in gene expression within populations. Our results suggest that evolved change in 5mC could contribute to phenotypic evolution by influencing variability in gene expression in invertebrates

Fluctuations in chromatin state at regulatory loci occur spontaneously under relaxed selection and are associated with epigenetically inherited variation in C. elegans gene expression

Some epigenetic information can be transmitted between generations without changes in the underlying DNA sequence. Changes in epigenetic regulators, termed epimutations, can occur spontaneously and be propagated in populations in a manner reminiscent of DNA mutations. Small RNA-based epimutations occur in C. elegans and persist for around 3–5 generations on average. Here, we explored whether chromatin states also undergo spontaneous change and whether this could be a potential alternative mechanism for transgenerational inheritance of gene expression changes. We compared the chromatin and gene expression profiles at matched time points from three independent lineages of C. elegans propagated at minimal population size. Spontaneous changes in chromatin occurred in around 1% of regulatory regions each generation. Some were heritable epimutations and were significantly enriched for heritable changes in expression of nearby protein-coding genes. Most chromatin-based epimutations were short-lived but a subset had longer duration. Genes subject to long-lived epimutations were enriched for multiple components of xenobiotic response pathways. This points to a possible role for epimutations in adaptation to environmental stressors

Tertiary siRNAs mediate paramutation in C. elegans.

In the nematode Caenorhabditis elegans, different small RNA-dependent gene silencing mechanisms act in the germline to initiate transgenerational gene silencing. Piwi-interacting RNAs (piRNAs) can initiate transposon and gene silencing by acting upstream of endogenous short interfering RNAs (siRNAs), which engage a nuclear RNA interference (RNAi) pathway to trigger transcriptional gene silencing. Once gene silencing has been established, it can be stably maintained over multiple generations without the requirement of the initial trigger and is also referred to as RNAe or paramutation. This heritable silencing depends on the integrity of the nuclear RNAi pathway. However, the exact mechanism by which silencing is maintained across generations is not understood. Here we demonstrate that silencing of piRNA targets involves the production of two distinct classes of small RNAs with different genetic requirements. The first class, secondary siRNAs, are localized close to the direct target site for piRNAs. Nuclear import of the secondary siRNAs by the Argonaute HRDE-1 leads to the production of a distinct class of small RNAs that map throughout the transcript, which we term tertiary siRNAs. Both classes of small RNAs are necessary for full repression of the target gene and can be maintained independently of the initial piRNA trigger. Consistently, we observed a form of paramutation associated with tertiary siRNAs. Once paramutated, a tertiary siRNA generating allele confers dominant silencing in the progeny regardless of its own transmission, suggesting germline-transmitted siRNAs are sufficient for multigenerational silencing. This work uncovers a multi-step siRNA amplification pathway that promotes germline integrity via epigenetic silencing of endogenous and invading genetic elements. In addition, the same pathway can be engaged in environmentally induced heritable gene silencing and could therefore promote the inheritance of acquired traits.This study was supported by funding from: Cancer Research UK (http://www.cancerresearchuk. org), grant RG57329 to EAM; European Research Council (erc.europa.eu/) Framework Programme 7, grant RG58558 to EAM; Gonville and Caius College fellowship to PS; Career Development Award from the Medical Research Council (http://www.mrc.ac.uk/) to PS. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).This is the final published version. It first appeared at http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005078