Dario Beraldi

G-quadruplex structures are stable and detectable in human genomic DNA.

The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome for transcriptional regulation, DNA replication and genome stability, but the nature and distribution of G-quadruplexes across the genome remains elusive. Here, we address the hypothesis that G-quadruplex structures exist within double-stranded genomic DNA and can be explicitly identified using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA.

Performance of Marker-Based Relatedness Estimators in Natural Populations of Outbred Vertebrates

Knowledge of relatedness between pairs of individuals plays an important role in many research areas including evolutionary biology, quantitative genetics, and conservation. Pairwise relatedness estimation methods based on genetic data from highly variable molecular markers are now used extensively as a substitute for pedigrees. Although the sampling variance of the estimators has been intensively studied for the most common simple genetic relationships, such as unrelated, half- and full-sib, or parent–offspring, little attention has been paid to the average performance of the estimators, by which we mean the performance across all pairs of individuals in a sample. Here we apply two measures to quantify the average performance: first, misclassification rates between pairs of genetic relationships and, second, the proportion of variance explained in the pairwise relatedness estimates by the true population relatedness composition (i.e., the frequencies of different relationships in the population). Using simulated data derived from exceptionally good quality marker and pedigree data from five long-term projects of natural populations, we demonstrate that the average performance depends mainly on the population relatedness composition and may be improved by the marker data quality only within the limits of the population relatedness composition. Our five examples of vertebrate breeding systems suggest that due to the remarkably low variance in relatedness across the population, marker-based estimates may often have low power to address research questions of interest.

Revealing the History of Sheep Domestication Using Retrovirus Integrations

Sheep retroviruses can be used to map the selective preferences of early farmers and trace livestock movements across Europe. Not Just Dinner on Legs Several thousand years ago, human beings realized the virtues of domesticating wild animals as easy meat. Soon other possibilities became apparent, and as revealed in a series of papers in this issue, early pastoralists became selective about breeding for wool, leather, milk, and muscle power. In two papers, Gibbs et al. report on the bovine genome sequence (p. 522; see the cover, the Perspective by Lewin, and the Policy Forum by Roberts) and trace the diversity and genetic history of cattle (p. 528), while Chessa et al. (p. 532) survey the occurrence of endogenous retroviruses in sheep and map their distribution to historical waves of human selection and dispersal across Europe. Finally, Ludwig et al. (p. 485) note the origins of variation in the coat-color of horses and suggest that it is most likely to have been selected for by humans in need of good-looking transport. The domestication of livestock represented a crucial step in human history. By using endogenous retroviruses as genetic markers, we found that sheep differentiated on the basis of their “retrotype” and morphological traits dispersed across Eurasia and Africa via separate migratory episodes. Relicts of the first migrations include the Mouflon, as well as breeds previously recognized as “primitive” on the basis of their morphology, such as the Orkney, Soay, and the Nordic short-tailed sheep now confined to the periphery of northwest Europe. A later migratory episode, involving sheep with improved production traits, shaped the great majority of present-day breeds. The ability to differentiate genetically primitive sheep from more modern breeds provides valuable insights into the history of sheep domestication.

Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

BackgroundMethylation of cytosine in DNA (5mC) is an important epigenetic mark that is involved in the regulation of genome function. During early embryonic development in mammals, the methylation landscape is dynamically reprogrammed in part through active demethylation. Recent advances have identified key players involved in active demethylation pathways, including oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) by the TET enzymes, and excision of 5fC by the base excision repair enzyme thymine DNA glycosylase (TDG). Here, we provide the first genome-wide map of 5fC in mouse embryonic stem (ES) cells and evaluate potential roles for 5fC in differentiation.ResultsOur method exploits the unique reactivity of 5fC for pulldown and high-throughput sequencing. Genome-wide mapping revealed 5fC enrichment in CpG islands (CGIs) of promoters and exons. CGI promoters in which 5fC was relatively more enriched than 5mC or 5hmC corresponded to transcriptionally active genes. Accordingly, 5fC-rich promoters had elevated H3K4me3 levels, associated with active transcription, and were frequently bound by RNA polymerase II. TDG down-regulation led to 5fC accumulation in CGIs in ES cells, which correlates with increased methylation in these genomic regions during differentiation of ES cells in wild-type and TDG knockout contexts.ConclusionsCollectively, our data suggest that 5fC plays a role in epigenetic reprogramming within specific genomic regions, which is controlled in part by TDG-mediated excision. Notably, 5fC excision in ES cells is necessary for the correct establishment of CGI methylation patterns during differentiation and hence for appropriate patterns of gene expression during development.

Gene mapping in the wild with SNPs: guidelines and future directions

One of the biggest challenges facing evolutionary biologists is to identify and understand loci that explain fitness variation in natural populations. This review describes how genetic (linkage) mapping with single nucleotide polymorphism (SNP) markers can lead to great progress in this area. Strategies for SNP discovery and SNP genotyping are described and an overview of how to model SNP genotype information in mapping studies is presented. Finally, the opportunity afforded by new generation sequencing and typing technologies to map fitness genes by genome-wide association studies is discussed.

Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC and 5hmC marks at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), both 5mC and 5hmC are read as cytosines and thus cannot be discriminated; however, in oxBS-seq, specific oxidation of 5hmC to 5-formylcytosine (5fC) and conversion of the newly formed 5fC to uracil (under bisulfite conditions) means that 5hmC can be discriminated from 5mC. A positive readout of actual 5mC is gained from a single oxBS-seq run, and 5hmC levels are inferred by comparison with a BS-seq run. Here we describe an optimized second-generation protocol that can be completed in 2 d.

Genome-wide association mapping identifies the genetic basis of discrete and quantitative variation in sexual weaponry in a wild sheep population

Understanding the genetic architecture of phenotypic variation in natural populations is a fundamental goal of evolutionary genetics. Wild Soay sheep (Ovis aries) have an inherited polymorphism for horn morphology in both sexes, controlled by a single autosomal locus, Horns. The majority of males have large normal horns, but a small number have vestigial, deformed horns, known as scurs; females have either normal horns, scurs or no horns (polled). Given that scurred males and polled females have reduced fitness within each sex, it is counterintuitive that the polymorphism persists within the population. Therefore, identifying the genetic basis of horn type will provide a vital foundation for understanding why the different morphs are maintained in the face of natural selection. We conducted a genome‐wide association study using ∼36 000 single nucleotide polymorphisms (SNPs) and determined the main candidate for Horns as RXFP2, an autosomal gene with a known involvement in determining primary sex characters in humans and mice. Evidence from additional SNPs in and around RXFP2 supports a new model of horn‐type inheritance in Soay sheep, and for the first time, sheep with the same horn phenotype but different underlying genotypes can be identified. In addition, RXFP2 was shown to be an additive quantitative trait locus (QTL) for horn size in normal‐horned males, accounting for up to 76% of additive genetic variation in this trait. This finding contrasts markedly from genome‐wide association studies of quantitative traits in humans and some model species, where it is often observed that mapped loci only explain a modest proportion of the overall genetic variation.

Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

Compelling evidence that a single nucleotide substitution in TYRP1 is responsible for coat-colour polymorphism in a free-living population of Soay sheep

Identifying the genes that underlie phenotypic variation in natural populations is a central objective of evolutionary genetics. Here, we report the identification of the gene and causal mutation underlying coat colour variation in a free-living population of Soay sheep (Ovis aries). We targeted tyrosinase-related protein 1 (TYRP1), a positional candidate gene based on previous work that mapped the Coat colour locus to an approximately 15 cM window on chromosome 2. We identified a non-synonymous substitution in exon IV that was perfectly associated with coat colour. This polymorphism is predicted to cause the loss of a cysteine residue that is highly evolutionarily conserved and likely to be of functional significance. We eliminated the possibility that this association is due to the presence of strong linkage disequilibrium with an unknown regulatory mutation by demonstrating that there is no difference in relative TYRP1 expression between colour morphs. Analysis of this putative causal mutation in a complex pedigree of more than 500 sheep revealed almost perfect co-segregation with coat colour (Χ2-test, p<0.0001, LOD=110.20), and very tight linkage between Coat colour and TYRP1 (LOD=29.50).

G-quadruplex structures mark human regulatory chromatin

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP–seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5′ UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor–induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.