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Transcription of tRNA genes by RNA Polymerase III (RNAPIII) is tightly regulated by signaling cascades in response to nutrient availability. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulatory factors have not been described. For that reason, we decoded the proteome of a single native tRNA gene locus in yeast. We observed dynamic reprogramming of the core RNAPIII transcription machinery upon nutrient perturbation. In addition, we identified Fpt1, a protein of unknown function. Fpt1 uniquely occupied tRNA genes but its occupancy varied and correlated with the efficiency of RNAPIII eviction upon nutrient perturbation. Decoding the proteome of a tRNA gene in the absence of Fpt1 revealed that Fpt1 promotes eviction of RNAPIII. Cells without Fpt1 also showed impaired shutdown of ribosome biogenesis genes upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and for tuning an integrated physiological response to changing metabolic demands.Abstract Figure

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template.

Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here we apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.

Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) is a powerful technique for mapping in vivo, genome-wide DNA–protein interactions. The interplay between DNA and proteins determines the transcriptional state of the genome. Using specific antibodies for the ChIP, it is possible to generate genome-wide profiles of histone posttranslational modifications, providing insight into the epigenetic memory and developmental potential of cells. The interactions between DNA and proteins involved in epigenetic regulation and transcription are highly dynamic during embryonic development. ChIP-seq allows for a detailed analysis of these dynamic changes in DNA–protein binding during embryogenesis. ChIP-seq is performed on protein epitopes that have been cross-linked to genomic DNA. After shearing the DNA, fragments bound by the (modified) protein of interest are captured with antibodies. The genomic loci of interest are identified by sequencing. Here, we provide a step-by-step ChIP-seq protocol that efficiently captures epitopes from relatively small embryo samples.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a ‘gate-keeper’ for the correct temporal and spatial development of the neural crest.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors., Highlighted article: A network of embryonic genes, many of which are expressed in the mesoderm and the organiser, can initiate transcription through a non-canonical mechanism.

Chromatin immunoprecipitation (ChIP) followed by deep sequencing (ChIP-seq) is a powerful technique for mapping in vivo, genome-wide DNA-protein interactions. The interplay between DNA and proteins determines the transcriptional state of the genome. Using specific antibodies for the ChIP, it is possible to generate genome-wide profiles of histone posttranslational modifications, providing insight into the epigenetic memory and developmental potential of cells. The interactions between DNA and proteins involved in epigenetic regulation and transcription are highly dynamic during embryonic development. ChIP-seq allows for a detailed analysis of these dynamic changes in DNA-protein binding during embryogenesis. ChIP-seq is performed on protein epitopes that have been cross-linked to genomic DNA. After shearing the DNA, fragments bound by the (modified) protein of interest are captured with antibodies. The genomic loci of interest are identified by sequencing. Here, we provide a step-by-step ChIP-seq protocol that efficiently captures epitopes from relatively small embryo samples.

Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein–DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus by DNA sequencing., Author summary DNA is packaged by proteins into a structure called chromatin. This packaging plays an important role in gene transcription, DNA replication, and DNA repair. The regulation of these processes is determined by the interplay of proteins that physically interact with specific loci. Although many chromatin proteins have been identified, it remains a challenge to comprehensively identify all the chromatin interactions at any specific locus by protein-based methods such as capture mass spectrometry (MS). Here, we take advantage of DNA barcoding technologies and high-throughput sequencing to tackle this problem. We developed Epi-Decoder, a method that identifies chromatin–protein interaction at a single-copy locus in budding yeast. Epi-Decoder was successfully applied at 3 different genomic loci, resulting in comprehensive overviews of local chromatin interactomes. Most factors identified are known to be involved in chromatin-associated processes such as transcription and DNA replication. We also identified unexpected chromatin–protein interactions. Furthermore, capturing the dynamic chromatin interactome by changing physiological conditions provided insight into the local response to stress. In conclusion, Epi-Decoder is an effective tool for measuring the local proteome of a specific genomic locus by DNA sequencing.

BackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. The most recent vertebrate genome duplication is that in Xenopus laevis, resulting from the hybridization of two closely related species about 17 million years ago [1]. However, little is known about the consequences of this duplication at the level of the genome, the epigenome and gene expression.ResultsOf the parental subgenomes, S chromosomes have degraded faster than L chromosomes ever since the genome duplication and until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination (NAHR) has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300 (Ep300)-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers are comprised of transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.ConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and NAHR, and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole genome duplication.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Bogdanovic, Ozren et al., The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases., Spanish and Andalusian government grants BFU2013-41322-P and BIO-396 to J.L.G.-S. supported this work. R.L. was supported by an Australian Research Council Future Fellowship (FT120100862) and a Sylvia and Charles Viertel Senior Medical Research Fellowship, and work in the laboratory of R.L. was funded by the Australian Research Council, National Health and Medical Research Council, and the Raine Medical Research Foundation. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962). The laboratory of M.V. is supported by grants from the Netherlands Organisation for Scientific Research (NWO-VIDI; 864.09.003) and Cancer Genomics Netherlands, a European Research Council starting grant (309384) and the European Union Framework Programme 7 Network of Excellence EpiGeneSys. J.R.E. was supported by the Gordon and Betty Moore Foundation (GBMF3034) and is an Investigator of the Howard Hughes Medical Institute. Work in the laboratory of M.M. is funded by grants from the Ministerio de Economia y Competitividad (BFU2011-23083), Comunidad Autónoma de Madrid (CELLDD-CM), and by the Pro-CNIC Foundation. This work has been supported by a grant from the US National Institutes of Health (National Institute of Child Health and Human Development, grant R01HD069344) to G.J.C.V.

Contains fulltext : 174204.pdf (Publisher’s version ) (Closed access) Transposable elements are parasitic genomic elements that can be deleterious for host gene function and genome integrity. Heterochromatic histone modifications are involved in the repression of transposons. However, it remains unknown how these histone modifications mark different types of transposons during embryonic development. Here we document the variety of heterochromatic epigenetic signatures at parasitic elements during development in Xenopus tropicalis, using genome-wide ChIP-sequencing data and ChIP-qPCR analysis. We show that specific subsets of transposons in various families and subfamilies are marked by different combinations of the heterochromatic histone modifications H4K20me3, H3K9me2/3 and H3K27me3. Many DNA transposons are marked at the blastula stage already, whereas at retrotransposons the histone modifications generally accumulate at the gastrula stage or later. Furthermore, transposons marked by H3K9me3 and H4K20me3 are more prominent in gene deserts. Using intra-subfamily divergence as a proxy for age, we show that relatively young DNA transposons are preferentially marked by early embryonic H4K20me3 and H3K27me3. In contrast, relatively young retrotransposons are marked by increasing H3K9me3 and H4K20me3 during development, and are also linked to piRNA-sized small non-coding RNAs. Our results implicate distinct repression mechanisms that operate in a transposon-selective and developmental stage-specific fashion.

Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences., Histone modifying enzymes are required for cell differentiation and lineage commitment during embryonic development. By a comprehensive set of epigenome reference maps of Xenopus embryos, the authors show that H3K4me3 and H3K27me3 exert an extended maternal control well into post-gastrulation development.

Polycomb group (PcG) proteins are key regulators in establishing a transcriptional repressive state. Polycomb Repressive Complex 2 (PRC2), one of the two major PcG protein complexes, is essential for proper differentiation and maintenance of cellular identity. Multiple factors are involved in recruiting PRC2 to its genomic targets. In this review, we will discuss the role of DNA sequence, transcription factors, pre-existing histone modifications, and RNA in guiding PRC2 towards specific genomic loci. The DNA sequence itself influences the DNA methylation state, which is an important determinant of PRC2 recruitment. Other histone modifications are also important for PRC2 binding as PRC2 can respond to different cellular states via crosstalk between histone modifications. Additionally, PRC2 might be able to sense the transcriptional status of genes by binding to nascent RNA, which could also guide the complex to chromatin. In this review, we will discuss how all these molecular aspects define a local chromatin state which controls accurate, cell-type-specific epigenetic silencing by PRC2.This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.

During embryonic development, maintenance of cell identity and lineage commitment requires the Polycomb-group PRC2 complex, which catalyzes histone H3 lysine 27 trimethylation (H3K27me3). However, the developmental origins of this regulation are unknown. Here we show that H3K27me3 enrichment increases from blastula stages onward in embryos of the Western clawed frog (Xenopus tropicalis) within constrained domains strictly defined by sequence. Strikingly, although PRC2 also 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 the basis of DNA sequence alone, with a sequence signature conserved between humans, frogs, and fish. These regions correspond to the subset of blastula-stage DNA methylation-free domains that are depleted for activating promoter motifs, and enriched for motifs of developmental factors. These results imply a genetic-default model in which a preexisting absence of DNA methylation is the major determinant of H3K27 methylation when not opposed by transcriptional activation. The sequence and motif signatures reveal the hierarchical and genetically inheritable features of epigenetic cross-talk that impose constraints on Polycomb regulation and guide H3K27 methylation during the exit of pluripotency.