Low-pass whole genome bisulfite sequencing of neonatal dried blood spots identifies a role for RUNX1 in Down syndrome DNA methylation profiles.

Neonatal dried blood spots (NDBS) are a widely banked sample source that enables retrospective investigation into early life molecular events. Here, we performed low-pass whole genome bisulfite sequencing (WGBS) of 86 NDBS DNA to examine early life Down syndrome (DS) DNA methylation profiles. DS represents an example of genetics shaping epigenetics, as multiple array-based studies have demonstrated that trisomy 21 is characterized by genome-wide alterations to DNA methylation. By assaying over 24 million CpG sites, thousands of genome-wide significant (q < 0.05) differentially methylated regions (DMRs) that distinguished DS from typical development and idiopathic developmental delay were identified. Machine learning feature selection refined these DMRs to 22 loci. The DS DMRs mapped to genes involved in neurodevelopment, metabolism, and transcriptional regulation. Based on comparisons with previous DS methylation studies and reference epigenomes, the hypermethylated DS DMRs were significantly (q < 0.05) enriched across tissues while the hypomethylated DS DMRs were significantly (q < 0.05) enriched for blood-specific chromatin states. A ~28 kb block of hypermethylation was observed on chromosome 21 in the RUNX1 locus, which encodes a hematopoietic transcription factor whose binding motif was the most significantly enriched (q < 0.05) overall and specifically within the hypomethylated DMRs. Finally, we also identified DMRs that distinguished DS NDBS based on the presence or absence of congenital heart disease (CHD). Together, these results not only demonstrate the utility of low-pass WGBS on NDBS samples for epigenome-wide association studies, but also provide new insights into the early life mechanisms of epigenomic dysregulation resulting from trisomy 21.

GC skew is a conserved property of unmethylated CpG island promoters across vertebrates.

GC skew is a measure of the strand asymmetry in the distribution of guanines and cytosines. GC skew favors R-loops, a type of three stranded nucleic acid structures that form upon annealing of an RNA strand to one strand of DNA, creating a persistent RNA:DNA hybrid. Previous studies show that GC skew is prevalent at thousands of human CpG island (CGI) promoters and transcription termination regions, which correspond to hotspots of R-loop formation. Here, we investigated the conservation of GC skew patterns in 60 sequenced chordates genomes. We report that GC skew is a conserved sequence characteristic of the CGI promoter class in vertebrates. Furthermore, we reveal that promoter GC skew peaks at the exon 1/ intron1 junction and that it is highly correlated with gene age and CGI promoter strength. Our data also show that GC skew is predictive of unmethylated CGI promoters in a range of vertebrate species and that it imparts significant DNA hypomethylation for promoters with intermediate CpG densities. Finally, we observed that terminal GC skew is conserved for a subset of vertebrate genes that tend to be located significantly closer to their downstream neighbors, consistent with a role for R-loop formation in transcription termination.

Holocentric chromosomes: convergent evolution, meiotic adaptations, and genomic analysis.

In most eukaryotes, the kinetochore protein complex assembles at a single locus termed the centromere to attach chromosomes to spindle microtubules. Holocentric chromosomes have the unusual property of attaching to spindle microtubules along their entire length. Our mechanistic understanding of holocentric chromosome function is derived largely from studies in the nematode Caenorhabditis elegans, but holocentric chromosomes are found over a broad range of animal and plant species. In this review, we describe how holocentricity may be identified through cytological and molecular methods. By surveying the diversity of organisms with holocentric chromosomes, we estimate that the trait has arisen at least 13 independent times (four times in plants and at least nine times in animals). Holocentric chromosomes have inherent problems in meiosis because bivalents can attach to spindles in a random fashion. Interestingly, there are several solutions that have evolved to allow accurate meiotic segregation of holocentric chromosomes. Lastly, we describe how extensive genome sequencing and experiments in nonmodel organisms may allow holocentric chromosomes to shed light on general principles of chromosome segregation.

Sorghum genome sequencing by methylation filtration.

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.

Rapid genome shrinkage in a self-fertile nematode reveals sperm competition proteins.

To reveal impacts of sexual mode on genome content, we compared chromosome-scale assemblies of the outcrossing nematode Caenorhabditis nigoni to its self-fertile sibling species, C. briggsaeC. nigoni's genome resembles that of outcrossing relatives but encodes 31% more protein-coding genes than C. briggsaeC. nigoni genes lacking C. briggsae orthologs were disproportionately small and male-biased in expression. These include the male secreted short (mss) gene family, which encodes sperm surface glycoproteins conserved only in outcrossing species. Sperm from mss-null males of outcrossing C. remanei failed to compete with wild-type sperm, despite normal fertility in noncompetitive mating. Restoring mss to C. briggsae males was sufficient to enhance sperm competitiveness. Thus, sex has a pervasive influence on genome content that can be used to identify sperm competition factors.

Autosomal Trisomy and Triploidy Are Corrected During Female Meiosis in Caenorhabditis elegans.

Trisomy and triploidy, defined as the presence of a third copy of one or all chromosomes, respectively, are deleterious in many species including humans. Previous studies have demonstrated that Caenorhabditis elegans with a third copy of the X chromosome are viable and fertile. However, the extra X chromosome was shown to preferentially segregate into the first polar body during oocyte meiosis to produce a higher frequency of euploid offspring than would be generated by random segregation. Here, we demonstrate that extra autosomes are preferentially eliminated by triploid C. elegans and trisomy IV C. elegans Live imaging of anaphase-lagging chromosomes and analysis of REC-8 staining of metaphase II spindles revealed that, in triploids, some univalent chromosomes do not lose cohesion and preferentially segregate intact into the first polar body during anaphase I, whereas other autosomes segregate chromatids equationally at anaphase I and eliminate some of the resulting single chromatids during anaphase II. We also demonstrate asymmetry in the anaphase spindle, which may contribute to the asymmetric segregation. This study reveals a pathway that allows aneuploid parents to produce euploid offspring at higher than random frequency.

Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products.

Metagenomics is a valuable tool for the study of microbial communities but has been limited by the difficulty of "binning" the resulting sequences into groups corresponding to the individual species and strains that constitute the community. Moreover, there are presently no methods to track the flow of mobile DNA elements such as plasmids through communities or to determine which of these are co-localized within the same cell. We address these limitations by applying Hi-C, a technology originally designed for the study of three-dimensional genome structure in eukaryotes, to measure the cellular co-localization of DNA sequences. We leveraged Hi-C data generated from a simple synthetic metagenome sample to accurately cluster metagenome assembly contigs into groups that contain nearly complete genomes of each species. The Hi-C data also reliably associated plasmids with the chromosomes of their host and with each other. We further demonstrated that Hi-C data provides a long-range signal of strain-specific genotypes, indicating such data may be useful for high-resolution genotyping of microbial populations. Our work demonstrates that Hi-C sequencing data provide valuable information for metagenome analyses that are not currently obtainable by other methods. This metagenomic Hi-C method could facilitate future studies of the fine-scale population structure of microbes, as well as studies of how antibiotic resistance plasmids (or other genetic elements) mobilize in microbial communities. The method is not limited to microbiology; the genetic architecture of other heterogeneous populations of cells could also be studied with this technique.

Applying word-based algorithms: the IMEter.

Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distinct from later introns and that these differences are closely related to the ability of some introns to increase gene expression. This word-based approach has proven more successful than deletion analysis at identifying the sequences responsible for elevating expression because they are dispersed throughout stimulatory introns.