Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos

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TBP2 is a substitute for TBP in Xenopus oocyte transcription

<p>Abstract</p> <p>Background</p> <p>TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos.</p> <p>Results</p> <p>We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription <it>in vivo</it>. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos.</p> <p>Conclusion</p> <p>The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation.</p

Temporal lack of DNA methylation-mediated repression is a universal feature of vertebrate development

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.-- et al.Peer Reviewe

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

Acknowledgements We thank Saartje Hontelez (Radboud University, Nijmegen), Sylvie Janssens and Kris Vleminckx (Vlaams Instituut voor Biotechnologie, Universiteit Gent) and Shelby Blythe (Princeton University) for advice on ChIP experiments; Caroline Hill (CRUK, LRI) for discussion on BMP signalling; Juan Larraín (Pontificia Universitad Católica de Chile) and Susan Fairley (European Bioinformatics Institute) for advice on RNA-seq experiments; Yvonne Turnbull (IMSARU, University of Aberdeen) for technical assistance; Alasdair MacKenzie (University of Aberdeen) for discussion and suggestions on the manuscript; Hajime Ogino (Nagahama Institute of Bio-Science and Technology) and Atsushi Suzuki (Hiroshima University) for plasmids; Pierre McCrea (University of Texas MD Anderson Cancer Center) for anti-Xenopus β-catenin antibody; The Genome Analysis Centre (TGAC, BBSRC, Norwich) for high-throughput sequencing; and Xenbase (http://www.xenbase.org) for reference database access. Funding This work was supported by the Biotechnology and Biological Sciences Research Council [BB/I003746/1 to S.H., BB/M001695/1 to S.H. and Y.N.]. Deposited in PMC for immediate release.Peer reviewedPublisher PD

GimmeMotifs: a de novo motif prediction pipeline for ChIP-sequencing experiments

Summary: Accurate prediction of transcription factor binding motifs that are enriched in a collection of sequences remains a computational challenge. Here we report on GimmeMotifs, a pipeline that incorporates an ensemble of computational tools to predict motifs de novo from ChIP-sequencing (ChIP-seq) data. Similar redundant motifs are compared using the weighted information content (WIC) similarity score and clustered using an iterative procedure. A comprehensive output report is generated with several different evaluation metrics to compare and evaluate the results. Benchmarks show that the method performs well on human and mouse ChIP-seq datasets. GimmeMotifs consists of a suite of command-line scripts that can be easily implemented in a ChIP-seq analysis pipeline

ChIP-Chip Designs to Interrogate the Genome of Xenopus Embryos for Transcription Factor Binding and Epigenetic Regulation

Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos. genome. In particular, a whole-genome microarray design was used to identify active promoters by close proximity to histone H3 lysine 4 trimethylation. A second microarray design features these experimentally derived promoter regions in addition to currently annotated 5′ ends of genes. These regions truly represent promoters as shown by binding of TBP, a key transcription initiation factor. embryos

Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date; after six months, it is available under a Creative Commons License.-- et al.The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the noncoding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here, we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that (1) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression, (2) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog, and (3) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation, suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage.We thank the Spanish and Andalusian Governments for grants (BFU2010-14839, CSD2007-00008, and Proyecto de Excelencia CVI-3488) for funding this study.Peer reviewe

WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality.V.B. and C.B. are funded by the Stand Up to Cancer campaign for Cancer Research UK, and Cancer Research UK Program Foundation Award to C.B. (C37275/1A20146). K.M. was supported by an NWO-VIDI grant (864.12.010)

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others