Project description:During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 K27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here, we report on H3K27me3 deposition dynamics in Xenopus embryos, on sequence elements that initiate deposition during pluripotency, and the sequence characteristics that segregate Polycomb-regulated domains from the rest of the genome. Strikingly, although PRC2 binds widely to active enhancers, H3K27me3 is only deposited at a small subset of these sites. Using a Support Vector Machine algorithm these sequences can be predicted accurately on basis of DNA sequence alone, with a sequence signature conserved between humans, frog and fish. The results suggest a genetic-default model in which genomic sequence constrains Polycomb regulation. ChIP-seq profiles of three histone modifications (H3K4me3, H3K27me3 and H3K4me1) and RNA Polymerase II, EZH2 and Jarid2 of Xenopus tropicalis embryos during development
Project description:Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.
Project description:Human oocyte cDNA library was hybridized on a multi-species oocyte array (Bovine, Mouse, Frog) Temperature stringency criteria was used to evaluate the conservation degree of oocyte genes among vertebrates (Bovine, Mouse, Frog)
Project description:During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 K27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here, we report on H3K27me3 deposition dynamics in Xenopus embryos, on sequence elements that initiate deposition during pluripotency, and the sequence characteristics that segregate Polycomb-regulated domains from the rest of the genome. Strikingly, although PRC2 binds widely to active enhancers, H3K27me3 is only deposited at a small subset of these sites. Using a Support Vector Machine algorithm these sequences can be predicted accurately on basis of DNA sequence alone, with a sequence signature conserved between humans, frog and fish. The results suggest a genetic-default model in which genomic sequence constrains Polycomb regulation.
Project description:Recent efforts towards the comprehensive identification of RNA-bound proteomes have revealed a large, surprisingly diverse family of candidate RNA-binding proteins (RBPs). Quantitative metrics for characterization and validation of protein-RNA interactions and their dynamic interactions have, however, proven to be analytically challenging and prone to error. Here we report a novel method termed LEAP-RBP for the selective, quantitative recovery of UV-crosslinked RNA-protein complexes. By virtue of its high specificity and yield, LEAP-RBP distinguishes RNA-bound and RNA-free protein levels and reveals common sources of experimental noise in RNA-centric RBP enrichment methods. We introduce new strategies for accurate RBP identification and signal-based metrics for quantifying protein-RNA complex enrichment, relative RNA occupancy, and method specificity. The utility of our approach is validated by comprehensive identification of RBPs whose association with mRNA is modulated in response to global mRNA translation state changes and through in-depth benchmark comparisons with current methodologies.