Genome Biology

Although chromosomal deletions and inversions are important in cancer, conventional methods for detecting DNA rearrangements require laborious indirect assays. Here we develop fluorescent reporters to rapidly quantify CRISPR/Cas9-mediated deletions and inversions. We find that inversion depends on the non-homologous end-joining enzyme LIG4. We also engineer deletions and inversions for a 50 kb Pten genomic region in mouse liver. We discover diverse yet sequence-specific indels at the rearrangement fusion sites. Moreover, we detect Cas9 cleavage at the fourth nucleotide on the non-complementary strand, leading to staggered instead of blunt DNA breaks. These reporters allow mechanisms of chromosomal rearrangements to be investigated.

Sociality has many rewards, but can also be dangerous, as high population density and low genetic diversity, common in social insects, is ideal for parasite transmission. Despite this risk, honeybees and other sequenced social insects have far fewer canonical immune genes relative to solitary insects. Social protection from infection, including behavioral responses, may explain this depauperate immune repertoire. Here, based on full genome sequences, we describe the immune repertoire of two ecologically and commercially important bumblebee species that diverged approximately 18 million years ago, the North American Bombus impatiens and European Bombus terrestris. We find that the immune systems of these bumblebees, two species of honeybee, and a solitary leafcutting bee, are strikingly similar. Transcriptional assays confirm the expression of many of these genes in an immunological context and more strongly in young queens than males, affirming Bateman’s principle of greater investment in female immunity. We find evidence of positive selection in genes encoding antiviral responses, components of the Toll and JAK/STAT pathways, and serine protease inhibitors in both social and solitary bees. Finally, we detect many genes across pathways that differ in selection between bumblebees and honeybees, or between the social and solitary clades. The similarity in immune complement across a gradient of sociality suggests that a reduced immune repertoire predates the evolution of sociality in bees. The differences in selection on immune genes likely reflect divergent pressures exerted by parasites across social contexts.

In mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. Here, we show a novel role for the lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, H3K27me3. Similar to Dxz4, Firre is X-linked and expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding sites, and is preferentially located adjacent to the nucleolus. CTCF binding present initially in both male and female mouse embryonic stem cells is lost from the active X during development. Knockdown of Firre disrupts perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating a role in maintenance of an important epigenetic feature of the inactive X chromosome. No X-linked gene reactivation is seen after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing is observed. Further experiments in female embryonic stem cells suggest that Firre does not play a role in X inactivation onset. The X-linked lncRNA Firre helps to position the inactive X chromosome near the nucleolus and to preserve one of its main epigenetic features.

Gene expression variation is a phenotypic trait of particular interest as it represents the initial link between genotype and other phenotypes. Analyzing how such variation apportions among and within groups allows for the evaluation of how genetic and environmental factors influence such traits. It also provides opportunities to identify genes and pathways that may have been influenced by non-neutral processes. Here we use a population genetics framework and next generation sequencing to evaluate how gene expression variation is apportioned among four human groups in a natural biological tissue, the placenta. We estimate that on average, 33.2%, 58.9%, and 7.8% of the placental transcriptome is explained by variation within individuals, among individuals, and among human groups, respectively. Additionally, when technical and biological traits are included in models of gene expression they each account for roughly 2% of total gene expression variation. Notably, the variation that is significantly different among groups is enriched in biological pathways associated with immune response, cell signaling, and metabolism. Many biological traits demonstrate correlated changes in expression in numerous pathways of potential interest to clinicians and evolutionary biologists. Finally, we estimate that the majority of the human placental transcriptome exhibits expression profiles consistent with neutrality; the remainder are consistent with stabilizing selection, directional selection, or diversifying selection. We apportion placental gene expression variation into individual, population, and biological trait factors and identify how each influence the transcriptome. Additionally, we advance methods to associate expression profiles with different forms of selection.