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Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

International audience; Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson–Gilford progeria syndrome (HGPS) and Rothmund–Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene (TP63) as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the TP63 gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of TP63. Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal TP63 RT as an early event in progeroid disease progression, and suggest TP63 gene regulation as a potential therapeutic target.

Several 400- to 800-kb murine chromosome domains switch from early to late replication during loss of pluripotency, accompanied by a stable form of gene silencing that is resistant to reprogramming. We found that, whereas enhanced nuclease accessibility correlated with early replication genome-wide, domains that switch replication timing during differentiation were exceptionally inaccessible even when early-replicating. Nonetheless, two domains studied in detail exhibited substantial changes in transcriptional activity and higher-order chromatin unfolding confined to the region of replication timing change. Chromosome conformation capture (4C) data revealed that in the unfolded state in embryonic stem cells, these domains interacted preferentially with the early-replicating chromatin compartment, rarely interacting even with flanking late-replicating domains, whereas after differentiation, these same domains preferentially associated with late-replicating chromatin, including flanking domains. In both configurations they retained local boundaries of self-interaction, supporting the replication domain model of replication-timing regulation. Our results reveal a principle of developmentally regulated, large-scale chromosome folding involving a subnuclear compartment switch of inaccessible chromatin. This unusual level of regulation may underlie resistance to reprogramming in replication-timing switch regions.

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

The replication timing of some genes is developmentally regulated, but the significance of replication timing to cellular differentiation has been difficult to substantiate. Studies have largely been restricted to the comparison of a few genes in established cell lines derived from different tissues, and most of these genes do not change replication timing. Hence, it has not been possible to predict how many or what types of genes might be subject to such control. Here, we have evaluated the replication timing of 54 tissue-specific genes in mouse embryonic stem cells before and after differentiation to neural precursors. Strikingly, genes residing within isochores rich in GC and poor in long interspersed nuclear elements (LINEs) did not change their replication timing, whereas half of genes within isochores rich in AT and long interspersed nuclear elements displayed programmed changes in replication timing that accompanied changes in gene expression. Our results provide direct evidence that differentiation-induced autosomal replication-timing changes are a significant part of mammalian development, provide a means to predict genes subject to such regulation, and suggest that replication timing may be more related to the evolution of metazoan genomes than to gene function or expression pattern.

We agree with Brunet and Doolittle (1) on the utility of distinguishing the evolutionarily selected effects (SE) of some genomic elements from the causal roles (CR) of other elements that lack signatures of selection (1⇓⇓–4). DNA sequences identified by biochemical approaches include both SE and CR elements, and genetic variation in both has been implicated in human traits and disease susceptibility. We thus view the Encyclopedia of DNA Elements (ENCODE) catalog and similar data resources as important foundations for understanding the DNA elements and molecular mechanisms underlying human biology and disease.

The chromosomal localization of the human and rat genes encoding the kainate-preferring glutamate receptor subunits KA1 and KA2 (GRIK4 and GRIK5, respectively) was determined by Southern analysis of rat x mouse and human x mouse somatic cell hybrid panels and by fluorescence in situ hybridization. The localization of the mouse genes (Grik4 and Grik5) was established by interspecific backcross mapping. GRIK4 and GRIK5 are located on separate chromosomes (Chrs) in all species. GRIK4 mapped to human Chr 11q22.3, mouse Chr 9, and rat Chr 8. GRIK5 mapped to human Chr 19q13.2, mouse Chr 7, and rat Chr 1. The genes encoding the (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-preferring subunit GluR4, or GluRD (GRIA4), the neural cell adhesion molecule (NCAM), the D2 dopamine receptor (DRD2), and the Thy-1 cell surface antigen (THY1) have all been previously mapped to the human Chr 11q22 region. The mapping of the human GRIK4 and GRIK5 genes confirms and extends the relationship between human Chr 11 and mouse Chr 9 and also human Chr 19 and mouse Chr 7. GRIK4 is the fifth gene shared by human Chr 11 and rat Chr 8, whereas GRIK5 is 1 out of the 12 genes that are located on both human Chr 19 and rat Chr 1. Our data extend the conserved synteny established between certain human, mouse, and rat Chrs.