TRES predicts transcription control in embryonic stem cells.

SUMMARY: Unraveling transcriptional circuits controlling embryonic stem cell maintenance and fate has great potential for improving our understanding of normal development as well as disease. To facilitate this, we have developed a novel web tool called 'TRES' that predicts the likely upstream regulators for a given gene list. This is achieved by integrating transcription factor (TF) binding events from 187 ChIP-sequencing and ChIP-on-chip datasets in murine and human embryonic stem (ES) cells with over 1000 mammalian TF sequence motifs. Using 114 TF perturbation gene sets, as well as 115 co-expression clusters in ES cells, we validate the utility of this approach. AVAILABILITY AND IMPLEMENTATION: TRES is freely available at http://www.tres.roslin.ed.ac.uk. CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.This work was supported by a University of Edinburgh Chancellors Fellowship awarded to AJ and strategic funding from the BBSRC. CP was funded by the Scottish Government through the Strategic Partnership for Animal Science Excellence (SPASE). The Gottgens’ lab is supported by LLR, the MRC, BBSRC, Cancer Research UK, and Wellcome Trust core support to the Cambridge Institute for Medical Research and Wellcome Trust–MRC Cambridge Stem Cell Institute.This version is the author accepted manuscript. The published advanced access version can be viewed on the journals website at: http://bioinformatics.oxfordjournals.org/content/early/2014/06/23/bioinformatics.btu399.full.pdf+htm

The phylogenetically distinct early human embryo

The phylogenetic singularity of the human embryo remains unresolved as cell types of the human blastocyst have resisted classification. Combining clustering of single cellular transcriptomes and dynamically expressed genes we resolve the cell types. This unveils the missing inner cell mass (ICM) and reveals classical step-wise development. Conversely, numerous features render our blastocyst phylogenetically distinct: unlike mice, our epiblast is self-renewing and we have blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. At the transcriptome-level all primate embryos are distinct as the pluripotent cell types are uniquely fast evolving. A substantial fraction of gene expression gain and loss events between human and new-world monkeys involve endogenous retrovirus H (ERVH). Human pluripotent cells are unique in which (H)ERVH's are active, the extent to which these modulate neighbour gene expression and their ability to suppress mutagenic transposable elements. Current naive cultures are heterogeneous and both developmentally and phylogenetically "confused"

Developmental Transcriptional Enhancers: A Subtle Interplay between Accessibility and Activity

Measurements of open chromatin in specific cell types are widely used to infer the spatiotemporal activity of transcriptional enhancers. How reliable are these predictions? In this review, it is argued that the relationship between the accessibility and activity of an enhancer is insufficiently described by simply considering open versus closed chromatin, or active versus inactive enhancers. Instead, recent studies focusing on the quantitative nature of accessibility signal reveal subtle differences between active enhancers and their different inactive counterparts: the closed silenced state and the accessible primed and repressed states. While the open structure as such is not a specific indicator of enhancer activity, active enhancers display a higher degree of accessibility than the primed and repressed states. Molecular mechanisms that may account for these quantitative differences are discussed. A model that relates molecular events at an enhancer to changes in its activity and accessibility in a developing tissue is also proposed

A theoretical model of Polycomb/Trithorax action unites stable epigenetic memory and dynamic regulation

Polycomb and Trithorax group proteins maintain stable epigenetic memory of gene expression states for some genes, but many targets show highly dynamic regulation. Here we combine experiment and theory to examine the mechanistic basis of these different modes of regulation. We present a mathematical model comprising a Polycomb/Trithorax response element (PRE/TRE) coupled to a promoter and including Drosophila developmental timing. The model accurately recapitulates published studies of PRE/TRE mediated epigenetic memory of both silencing and activation. With minimal parameter changes, the same model can also recapitulate experimental data for a different PRE/TRE that allows dynamic regulation of its target gene. The model predicts that both cell cycle length and PRE/TRE identity are critical for determining whether the system gives stable memory or dynamic regulation. Our work provides a simple unifying framework for a rich repertoire of PRE/TRE functions, and thus provides insights into genome-wide Polycomb/Trithorax regulation.Peer Reviewe

Doctor of Philosophy

dissertationOne central question in development is how totipotency and pluripotency are established. In mature human sperm, genes of importance for embryo development (i.e. transcription factors) lack DNA methylation and bear nucleosomes with distinctive histone modifications, suggesting the specialized packaging of these developmental genes in the germline. Here, we explored the tractable zebrafish model and found conceptual conservation as well as several new features. Biochemical and mass spectrometric approaches reveal the zebrafish sperm genome packaged in nucleosomes and histone variants (and not protamine), and we find linker histones high and H4K16ac absent-key factors which may contribute to genome condensation. We examined several activating (H3K4me2/3, H3K14ac, H2AFV) and repressing (H3K27me3, H3K36me3, H3K9me3, hypoacetylation) modifications/compositions genome-wide, and find developmental genes packaged in large blocks of chromatin with coincident activating and repressing marks and DNA hypomethylation, revealing complex "multivalent" chromatin. Notably, genes that acquire DNA methylation in the soma (muscle) are enriched in transcription factors for alternative cell fates. Remarkably, we find H3K36me3 located in "silent" developmental gene promoters, and not present at the 3' ends of coding regions of genes heavily transcribed during sperm maturation, suggesting different rules for H3K36me3 in iv the germline and soma. We also reveal the chromatin patterns of transposons, rDNA, and tRNAs. Finally, high levels of H3K4me3 and H3K14ac in sperm are correlated with genes activated in embryos prior to the mid-blastula transition (MBT), whereas multivalent genes are correlated with activation at or after MBT. Taken together, gene sets with particular functions in the embryo are packaged by distinctive types of complex and often atypical chromatin in sperm. Bivalent marks, as the chromatin signature of pluripotency, are not persistent and diluted during early synchronous cell division, making them arguable to be heritable epigenetic marks. Studies in early embryos indicate DNA methylation status is the fundamental to confer totipotency and pluripotency. The anticorrelation between DNA methylation profiles and H2A.Z occupancy is conserved from plants to vertebrates. Here, we examined H2afva occupancy in early embryos in zebrafish by ChIP-seq. We found both H2afva level and enrichment remain consistent from sperm to embryos. H2afva is enriched in proximal promoter region in the first nucleosome. Consistent with previous studies, H2afva occupancy is anticorrelated to DNA methylation both in the promoters and outside of promoters. These data suggest H2afva is potentially a heritable epigenetic mark and sets up DNA methylation profiles of totipotency and pluripotency

Gene expression variability in mammalian embryonic stem cells using single cell RNA-seq data

AbstractBackgroundGene expression heterogeneity contributes to development as well as disease progression. Due to technological limitations, most studies to date have focused on differences in mean expression across experimental conditions, rather than differences in gene expression variance. The advent of single cell RNA sequencing has now made it feasible to study gene expression heterogeneity and to characterise genes based on their coefficient of variation.MethodsWe collected single cell gene expression profiles for 32 human and 39 mouse embryonic stem cells and studied correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further systematically characterised properties unique to High CV genes.ResultsHighly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent (H3K4me3 and H3K27me3) marked promoters and showed enrichment for response to DNA damage and DNA repair.ConclusionsTaken together, this analysis demonstrates the divergent characteristics of genes based on their CV. High CV genes tend to form co-expression clusters and they explain bivalency at least in part

Efecto del origen y las condiciones de cultivo en la heterogeneidad de poblaciones celulares pluripotentes

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Fisiología Animal, leída el 04-07-2014Sección Deptal. de Fisiología (Veterinaria)Fac. de VeterinariaTRUEunpu

Theoretical analysis of Polycomb-Trithorax systems predicts that poised chromatin is bistable and not bivalent

Polycomb and Trithorax group proteins regulate silent and active gene expression states, but also allow poised states in pluripotent cells. Here the authors present a mathematical model that integrates data on Polycomb/ Trithorax biochemistry into a single coherent framework which predicts that poised chromatin is not bivalent as previously proposed, but is bistable, meaning that the system switches frequently between stable active and silent states

Multiple roles of glyoxalase 1-mediated suppression of methylglyoxal glycation in cancer biology—involvement in tumour suppression, tumour growth, multidrug resistance and target for chemotherapy

Glyoxalase 1 (Glo1) is part of the glyoxalase system in the cytoplasm of all human cells. It catalyses the glutathione-dependent removal of the endogenous reactive dicarbonyl metabolite, methylglyoxal (MG). MG is formed mainly as a side product of anaerobic glycolysis. It modifies protein and DNA to form mainly hydroimidazolone MG-H1 and imidazopurinone MGdG adducts, respectively. Abnormal accumulation of MG, dicarbonyl stress, increases adduct levels which may induce apoptosis and replication catastrophe. In the non-malignant state, Glo1 is a tumour suppressor protein and small molecule inducers of Glo1 expression may find use in cancer prevention. Increased Glo1 expression is permissive for growth of tumours with high glycolytic activity and is thereby a biomarker of tumour growth. High Glo1 expression is a cause of multi-drug resistance. It is produced by over-activation of the Nrf2 pathway and GLO1 amplification. Glo1 inhibitors are antitumour agents, inducing apoptosis and necrosis, and anoikis. Tumour stem cells and tumours with high flux of MG formation and Glo1 expression are sensitive to Glo1 inhibitor therapy. It is likely that MG-induced cell death contributes to the mechanism of action of current antitumour agents. Common refractory tumours have high prevalence of Glo1 overexpression for which Glo1 inhibitors may improve therapy

Identifying novel regulators of human pluripotency and embryogenesis

In preimplantation development, the pluripotent epiblast is the precursor of all foetal tissues and of human embryonic stem cells (hESCs). Investigating the regulatory mechanisms that underpin human pluripotency is therefore vital to understand human embryogenesis and to improve in vitro models of human pluripotency. However, our current knowledge of the transcriptional regulation of human pluripotency is incomplete. In particular, various regulators identified through studies in the mouse display non-conserved expression or function in the human embryo. To explore how such proteins might be involved in the regulation of human pluripotency, I identified a number of transcription factors that are enriched in the human epiblast and expressed specifically in hESCs cultured under naïve, but not primed, pluripotency conditions. Through analysis of expression in both naïve hESCs and the developing human blastocyst, I determined that KLF17 is a promising candidate pluripotency regulator. I therefore performed gain- and loss-of-function analyses to elucidate the function of KLF17 in hESCs. Through ectopic expression of KLF17, I found that it is sufficient to upregulate the expression of a number of naïve hESC-associated genes in primed conditions and to drive transgene-mediated resetting of primed to naïve pluripotency under appropriate culture conditions. However, a CRISPR-Cas9-mediated null mutation of KLF17 revealed that it is not required for naïve pluripotency acquisition or maintenance in vitro. By transcriptome analysis of KLF17-null mutant hESCs during resetting, I identified possible compensatory mechanisms including upregulated expression of paralogous genes and the impact of exogenous WNT inhibition. In all, this work shows a role for KLF17 in establishing naïve pluripotency, but that it is not strictly necessary for generating naïve hESCs. I therefore suggest that the function of KLF17 is to promote a naïve pluripotent phenotype but that under standard conditions, parallel mechanisms exist and are able to compensate in the absence of KLF17