Selfish genetic elements and male fertility.

Selfish genetic elements (SGEs) are diverse and near ubiquitous in Eukaryotes and can be potent drivers of evolution. Here, we discuss SGEs that specifically act on sperm to gain a transmission advantage to the next generation. The diverse SGEs that affect sperm often impose costs on carrier males, including damaging ejaculates, skewing offspring sex ratios and in particular reducing sperm-competitive success of SGE-carrying males. How males and females tolerate and mitigate against these costs is a dynamic and expanding area of research. The intense intra-genomic conflict that these selfish elements generate could also have implications for male fertility and spermatogenesis more widely. This article is part of the theme issue 'Fifty years of sperm competition'.

Repetitive Elements Contribute to the Diversity and Evolution of Centromeres in the Fungal Genus Verticillium.

Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the 10 species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150-kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen Verticillium dahliae Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated long terminal repeat (LTR) retrotransposon is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus VerticilliumIMPORTANCE The genus Verticillium contains 10 species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization, and rapid evolution of centromeres in a set of closely related fungal species.

MtDNA sequence features associated with 'selfish genomes' predict tissue-specific segregation and reversion.

Mitochondrial DNA (mtDNA) encodes cellular machinery vital for cell and organism survival. Mutations, genetic manipulation, and gene therapies may produce cells where different types of mtDNA coexist in admixed populations. In these admixtures, one mtDNA type is often observed to proliferate over another, with different types dominating in different tissues. This 'segregation bias' is a long-standing biological mystery that may pose challenges to modern mtDNA disease therapies, leading to substantial recent attention in biological and medical circles. Here, we show how an mtDNA sequence's balance between replication and transcription, corresponding to molecular 'selfishness', in conjunction with cellular selection, can potentially modulate segregation bias. We combine a new replication-transcription-selection (RTS) model with a meta-analysis of existing data to show that this simple theory predicts complex tissue-specific patterns of segregation in mouse experiments, and reversion in human stem cells. We propose the stability of G-quadruplexes in the mtDNA control region, influencing the balance between transcription and replication primer formation, as a potential molecular mechanism governing this balance. Linking mtDNA sequence features, through this molecular mechanism, to cellular population dynamics, we use sequence data to obtain and verify the sequence-specific predictions from this hypothesis on segregation behaviour in mouse and human mtDNA.

H1 linker histones silence repetitive elements by promoting both histone H3K9 methylation and chromatin compaction.

Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.

A selfish genetic element linked to increased lifespan impacts metabolism in female house mice.

Gene drive systems can lead to the evolution of traits that further enhance the transmission of the driving element. In gene drive, one allele is transmitted to offspring at a higher frequency than the homologous allele. This has a range of consequences, which generally include a reduction in fitness of the carrier of the driving allele, making such systems 'selfish'. The t haplotype is one such driver, found in house mice. It is linked to a reduction in litter size in matings among heterozygous animals, but also to increased lifespan in wild females that carry it. Here, we tested whether carrying the t haplotype was associated with altered resting metabolic rate (RMR). We show that females carrying the t haplotype decrease RMR as they increase in size, compared with wild-type females or males of either genotype. Our study elucidates a plausible mechanism by which a selfish genetic element increases lifespan.

Special Issue: Repetitive DNA Sequences.

Repetitive DNAs are ubiquitous in eukaryotic genomes and, in many species, comprise the bulk of the genome. Repeats include transposable elements that can self-mobilize and disperse around the genome and tandemly-repeated satellite DNAs that increase in copy number due to replication slippage and unequal crossing over. Despite their abundance, repetitive DNAs are often ignored in genomic studies due to technical challenges in identifying, assembling, and quantifying them. New technologies and methods are now allowing unprecedented power to analyze repetitive DNAs across diverse taxa. Repetitive DNAs are of particular interest because they can represent distinct modes of genome evolution. Some repetitive DNAs form essential genome structures, such as telomeres and centromeres, that are required for proper chromosome maintenance and segregation, while others form piRNA clusters that regulate transposable elements; thus, these elements are expected to evolve under purifying selection. In contrast, other repeats evolve selfishly and cause genetic conflicts with their host species that drive adaptive evolution of host defense systems. However, the majority of repeats likely accumulate in eukaryotes in the absence of selection due to mechanisms of transposition and unequal crossing over. However, even these "neutral" repeats may indirectly influence genome evolution as they reach high abundance. In this Special Issue, the contributing authors explore these questions from a range of perspectives.

Carrying a selfish genetic element predicts increased migration propensity in free-living wild house mice.

Life is built on cooperation between genes, which makes it vulnerable to parasitism. Selfish genetic elements that exploit this cooperation can achieve large fitness gains by increasing their transmission relative to the rest of the genome. This leads to counter-adaptations that generate unique selection pressures on the selfish genetic element. This arms race is similar to host-parasite coevolution, as some multi-host parasites alter the host's behaviour to increase the chance of transmission to the next host. Here, we ask if, similarly to these parasites, a selfish genetic element in house mice, the t haplotype, also manipulates host behaviour, specifically the host's migration propensity. Variants of the t that manipulate migration propensity could increase in fitness in a meta-population. We show that juvenile mice carrying the t haplotype were more likely to emigrate from and were more often found as migrants within a long-term free-living house mouse population. This result may have applied relevance as the t has been proposed as a basis for artificial gene drive systems for use in population control.

Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral 'pathogens' cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.

Novel duplication pattern of the mitochondrial control region in Cantor's Giant softshell turtle Pelochelys cantorii.

Cantor's Giant Softshell Turtle, Pelochelys cantorii has become one of the most critically endangered species in the world. When comparative analyses of the P. cantorii complete mitochondrial genome sequences were conducted, we discovered a duplication of a segment of the control region in the mitochondrial genome of P. cantorii. The duplication is characterized by two copies of conserved sequence box 2 (CSB2) and CSB3 in a single control region. In contrast to previous reports of duplications involving the control regions of other animals, this particular pattern of duplications appears to be unique to P. cantorii. Copies of the CSB2 and CSB3 show many of the conserved sequence features typically found in mitochondrial control regions, and rare differences were found between the paralogous copies. Using the primer design principle of simple sequence repeats (SSR) and the reference sequence of the duplicated CSBs, specific primers were designed to amplify the duplicated CSBs. These primers were validated among different individuals and populations of P. cantorii. This unique duplication structure suggests the two copies of the CSB2 and CSB3 may have arisen through occasional tandem duplication and subsequent concerted evolution.

Unique Length-Dependent Biophysical Properties of Repetitive DNA.

Expansion of a trinucleotide repeat (TNR) sequence is the molecular signature of several neurological disorders. The formation of noncanonical structures by the TNR sequence is proposed to contribute to the expansion mechanism. Furthermore, it is known that the propensity for expansion increases with repeat length. In this work, we use calorimetry to describe the thermodynamic parameters (ΔH, TΔS, and ΔG) of the noncanonical stem-loop hairpins formed by the TNR sequences (CAG)n and (CTG)n, as well as the canonical (CAG)n/(CTG)n duplexes, for n = 6-14. Using a thermodynamic cycle, we calculated the same thermodynamic parameters describing the process of converting from noncanonical stem-loop hairpins to a canonical duplex. In addition to these thermodynamic analyses, we used spectroscopic techniques to determine the rate at which the noncanonical structures convert to duplex and the activation enthalpy ΔH(⧧) describing this process. We report that the thermodynamic parameters of unfolding the stem-loop (CTG)n and (CAG)n hairpins, along with the thermodynamic and kinetic properties of hairpin to duplex conversion, do not proportionally correspond to the increase in length, but rather show a unique pattern that depends on whether the sequence has an even or odd number of repeats.

First complete mitochondrial genome of primitive crab Homologenus malayensis (Decapoda: Brachyura: Podotremata: Homolidae).

The taxonomy and phylogeny of brachyuran crabs, and particularly of the Podotremata, have been the subjects of controversy due to their morphological diversity and complexity. The first complete mitochondrial genome (mitogenome) sequence of primitive crab Homologenus malayensis has been determined. The mitogenome is 15,793 bp in length, with A + T content 71.7%. The gene content and order are consistent with those in typical brachyuran crabs. A putative control region of 883 bp is identified due to its position (between srRNA and tRNA(Ile)) and AT richness (75.5%). Notably, the control region in H. malayensis contains nine identical specific repeat units of 42 bp and 11 identical repeat units of 2 bp with a total length of 400 bp, which is different from other crabs. These results are expected to provide useful information on both genomics and the future phylogenetic study of primitive crabs.

Complete sequence and gene organization of the mitochondrial genome of Podocnemis unifilis (Pleurodira: Podocnemididae).

The mitochondrial genome of Podocnemis unifilis (Pleurodira: Podocnemididae) is 16,493 bp in length, which contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The A+T content of the overall base composition of H-strand is 61.7% (T: 27.7%, C: 25.5%, A: 34.0%, G: 12.8%). ATP6, ATP8, CO3, ND4-6 and Cyt b genes begin with ATG as start codon, ND1, ND2 and ND4L begin with ATT, CO1 starts with GTG, CO2 starts with ATA, ND3 starts with ATC. ATP6, CO2, ND4L and ND5 genes are terminated with TAA as stop codon, ND1, ND2, ND3, ND6 and CO1 end with TAG, and the other four protein-coding genes end with an incomplete stop codon (a single stop nucleotide T or TA). A long unit repeat region is found in the control region.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons.

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

A repetitive DNA element regulates expression of the Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism.

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

The differential processing of telomeres in response to increased telomeric transcription and RNA-DNA hybrid accumulation.

Telomeres are protective nucleoprotein structures at the ends of eukaryotic chromosomes. Despite the heterochromatic state of telomeres they are transcribed, generating non-coding telomeric repeat-containing RNA (TERRA). Strongly induced TERRA transcription has been shown to cause telomere shortening and accelerated senescence in the absence of both telomerase and homology-directed repair (HDR). Moreover, it has recently been demonstrated that TERRA forms RNA-DNA hybrids at chromosome ends. The accumulation of RNA-DNA hybrids at telomeres also leads to rapid senescence and telomere loss in the absence of telomerase and HDR. Conversely, in the presence of HDR, telomeric RNA-DNA hybrid accumulation and increased telomere transcription promote telomere recombination, and hence, delayed senescence. Here, we demonstrate that despite these similar phenotypic outcomes, telomeres that are highly transcribed are not processed in the same manner as those that accumulate RNA-DNA hybrids.

HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment.

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase-telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA-protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation.

hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations.

Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.

Functions of DNA methylation: islands, start sites, gene bodies and beyond.

DNA methylation is frequently described as a 'silencing' epigenetic mark, and indeed this function of 5-methylcytosine was originally proposed in the 1970s. Now, thanks to improved genome-scale mapping of methylation, we can evaluate DNA methylation in different genomic contexts: transcriptional start sites with or without CpG islands, in gene bodies, at regulatory elements and at repeat sequences. The emerging picture is that the function of DNA methylation seems to vary with context, and the relationship between DNA methylation and transcription is more nuanced than we realized at first. Improving our understanding of the functions of DNA methylation is necessary for interpreting changes in this mark that are observed in diseases such as cancer.

Combination of de novo assembly of massive sequencing reads with classical repeat prediction improves identification of repetitive sequences in Schistosoma mansoni.

The genome of the parasitic platyhelminth Schistosoma mansoni is composed of approximately 40% of repetitive sequences of which roughly 20% correspond to transposable elements. When the genome sequence became available, conventional repeat prediction programs were used to find these repeats, but only a fraction could be identified. To exhaustively characterize the repeats we applied a new massive sequencing based strategy: we re-sequenced the genome by next generation sequencing, aligned the sequencing reads to the genome and assembled all multiple-hit reads into contigs corresponding to the repetitive part of the genome. We present here, for the first time, this de novo repeat assembly strategy and we confirm that such assembly is feasible. We identified and annotated 4,143 new repeats in the S. mansoni genome. At least one third of the repeats are transcribed. This strategy allowed us also to identify 14 new microsatellite markers, which can be used for pedigree studies. Annotations and the combined (previously known and new) 5,420 repeat sequences (corresponding to 47% of the genome) are available for download (http://methdb.univ-perp.fr/downloads/).

Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation.

Some herpesviruses, particularly lymphotropic viruses such as Marek's disease virus (MDV) and human herpesvirus 6 (HHV-6), integrate their DNA into host chromosomes. MDV and HHV-6, among other herpesviruses, harbor telomeric repeats (TMRs) identical to host telomeres at either end of their linear genomes. Using MDV as a natural virus-host model, we show that herpesvirus TMRs facilitate viral genome integration into host telomeres and that integration is important for establishment of latency and lymphoma formation. Integration into host telomeres also aids in reactivation from the quiescent state of infection. Our results and the presence of TMRs in many herpesviruses suggest that integration mediated by viral TMRs is a conserved mechanism, which ensures faithful virus genome maintenance in host cells during cell division and allows efficient mobilization of dormant viral genomes. This finding is of particular importance as reactivation is critical for virus spread between susceptible individuals and is necessary for continued herpesvirus evolution and survival.