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Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a “cut and paste” transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430 bp) element, while overexpression inhibition was observed for a larger (7,126 bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes. 

Abstract DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability 1,2 . At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs) 3–6 , subTADs 7 and loops 8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase. 

Abstract Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity. 

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.