Activin/nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark.

Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin-SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs.We thank Andrew Knights for the technical support and helpful discussion, and the Wellcome-Trust Sanger Institute Microarray and Next-Generation Sequencing facilities for the technical support. We also thank the Sanger Institute Mouse Genetics Projects for mouse production and genotyping. This work was supported by the European Research Council starting grant Relieve-IMDs and the Cambridge Hospitals National Institute for Health Research Biomedical Research Centre (L.V.), a British Heart Foundation Ph.D. Studentship (A.B.), a Federation of European Biochemical Societies (FEBS) long-term fellowship and EU Fp7 grant InnovaLIV (S.P.), EU Fp7 grant TissuGEN (S.M.), and Wellcome Trust grant 098051 (D.G.). A.B. conceived the research, performed and analyzed the experiments, and wrote the manuscript. P.M. computationally analyzed ChIP-seq data sets and performed statistical analyses. N.C.H., S.B., and R.A.P. provided technical support. A.G. performed embryo dissections and dysmorphology assessments. I.M. and D.B. performed teratoma assays. D.G. supervised the bioinformatics data analysis. S.P., S.M., and L.V. conceived the research and wrote the manuscript.This is the final published version. It first appeared at http://genesdev.cshlp.org/content/29/7/702.full

Evaluation of the chicken transcriptome by SAGE of B cells and the DT40 cell line

BACKGROUND: The understanding of whole genome sequences in higher eukaryotes depends to a large degree on the reliable definition of transcription units including exon/intron structures, translated open reading frames (ORFs) and flanking untranslated regions. The best currently available chicken transcript catalog is the Ensembl build based on the mappings of a relatively small number of full length cDNAs and ESTs to the genome as well as genome sequence derived in silico gene predictions. RESULTS: We use Long Serial Analysis of Gene Expression (LongSAGE) in bursal lymphocytes and the DT40 cell line to verify the quality and completeness of the annotated transcripts. 53.6% of the more than 38,000 unique SAGE tags (unitags) match to full length bursal cDNAs, the Ensembl transcript build or the genome sequence. The majority of all matching unitags show single matches to the genome, but no matches to the genome derived Ensembl transcript build. Nevertheless, most of these tags map close to the 3' boundaries of annotated Ensembl transcripts. CONCLUSIONS: These results suggests that rather few genes are missing in the current Ensembl chicken transcript build, but that the 3' ends of many transcripts may not have been accurately predicted. The tags with no match in the transcript sequences can now be used to improve gene predictions, pinpoint the genomic location of entirely missed transcripts and optimize the accuracy of gene finder software

The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency.

The TGFβ pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer.We thank Cambridge Genomic Services for help in next generation sequencing. The work was 203 supported by the European Research Council starting grant “Relieve IMDs” (L.V., S.B., A.B., 204 P.M.); the Cambridge University Hospitals National Institute for Health Research Biomedical 205 Research Center (L.V., J.K., A.S.L.); the Wellcome Trust PhD program (A.O., L.Y.); a British 206 Heart Foundation PhD studentship (FS/11/77/39327 to A.B.); a Grant-in-Aid for JSPS Fellows 207 (16J08005 to S.N.); and a core support grant from the Wellcome Trust and Medical Research 208 Council to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions

QUBIC, a specific and highly sensitive method for detection of protein–protein interactions, is used to identify new partners for the mitotic spindle components pericentrin and TACC3

RAD21 Cooperates with Pluripotency Transcription Factors in the Maintenance of Embryonic Stem Cell Identity

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21 - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program

Methodical Approach for Designing Electric Propulsion Systems Containing Two Motors

To reduce the environmental impact of the petroleum-based transport, new safe, cost and energy optimized electric vehicle propulsion concepts are mandatory. Besides the activities for improving the battery and power electronics technologies, highly integrated units and new functional component operation strategies are of special interest. In this paper, a methodical approach for deriving electric motor design values for electric propulsion systems, especially usable for urban vehicles, including two electric machines, is shown. Considering the overall results a new highly integrated electric drive containing two different sized electric motors, two clutches and a compressor in one single unit is used as an example. The design led to a variety of functional motor modes presented in the paper. On upscaled measurement results, the efficiency tables for 50 kW and 10 kW PSM- and ASM–motors are used for a combined working strategy. Using the 10 kW motor on low torque and speed requests the traction energy demand of a urban vehicle could be reduced in the range up to 5% on several drive cycles

A Quantitative Proteomics Tool To Identify DNA–Protein Interactions in Primary Cells or Blood

Interactions between transcription factors and genomic DNA, and in particular their impact on disease and cell fate, have been extensively studied on a global level using techniques based on next-generation sequencing. These approaches, however, do not allow an unbiased study of protein complexes that bind to certain DNA sequences. DNA pulldowns from crude lysates combined with quantitative mass spectrometry were recently introduced to close this gap. Established protocols, however, are restricted to cell lines because they are based on metabolic labeling or require large amounts of material. We introduce a high-throughput-compatible DNA pulldown that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate that our method can efficiently identify transcription factors binding to their consensus DNA motifs in extracts from primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. Nuclear proteomes with absolute quantification of nearly 7000 proteins in K562 cells and PBMCs clearly link differential interactions to differences in protein abundance, hence stressing the importance of selecting relevant cell extracts for any interaction in question. As shown for rs6904029, a SNP highly associated with chronic lymphocytic leukemia, our approach can provide invaluable functional data, for example, through integration with GWAS

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins

Embryonic stem [ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTO-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks.close17617