Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility.

OBJECTIVE: Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants. METHODS: We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression. RESULTS: We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity. SIGNIFICANCE: New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

Widespread transcriptional pausing and elongation control at enhancers.

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

Genomes of the Mouse Collaborative Cross.

The Collaborative Cross (CC) is a multiparent panel of recombinant inbred (RI) mouse strains derived from eight founder laboratory strains. RI panels are popular because of their long-term genetic stability, which enhances reproducibility and integration of data collected across time and conditions. Characterization of their genomes can be a community effort, reducing the burden on individual users. Here we present the genomes of the CC strains using two complementary approaches as a resource to improve power and interpretation of genetic experiments. Our study also provides a cautionary tale regarding the limitations imposed by such basic biological processes as mutation and selection. A distinct advantage of inbred panels is that genotyping only needs to be performed on the panel, not on each individual mouse. The initial CC genome data were haplotype reconstructions based on dense genotyping of the most recent common ancestors (MRCAs) of each strain followed by imputation from the genome sequence of the corresponding founder inbred strain. The MRCA resource captured segregating regions in strains that were not fully inbred, but it had limited resolution in the transition regions between founder haplotypes, and there was uncertainty about founder assignment in regions of limited diversity. Here we report the whole genome sequence of 69 CC strains generated by paired-end short reads at 30× coverage of a single male per strain. Sequencing leads to a substantial improvement in the fine structure and completeness of the genomes of the CC. Both MRCAs and sequenced samples show a significant reduction in the genome-wide haplotype frequencies from two wild-derived strains, CAST/EiJ and PWK/PhJ. In addition, analysis of the evolution of the patterns of heterozygosity indicates that selection against three wild-derived founder strains played a significant role in shaping the genomes of the CC. The sequencing resource provides the first description of tens of thousands of new genetic variants introduced by mutation and drift in the CC genomes. We estimate that new SNP mutations are accumulating in each CC strain at a rate of 2.4 ± 0.4 per gigabase per generation. The fixation of new mutations by genetic drift has introduced thousands of new variants into the CC strains. The majority of these mutations are novel compared to currently sequenced laboratory stocks and wild mice, and some are predicted to alter gene function. Approximately one-third of the CC inbred strains have acquired large deletions (>10 kb) many of which overlap known coding genes and functional elements. The sequence of these mice is a critical resource to CC users, increases threefold the number of mouse inbred strain genomes available publicly, and provides insight into the effect of mutation and drift on common resources.

Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks.

The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass.

Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X.

Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation.

New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gain-of-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis.