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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.

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.

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.

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.

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.

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.