Genome-scale transcriptomic and epigenomic analysis of stem cells

Embryonic stem cells (ESCs) are a special type of cell marked by two key properties: The capacity to create an unlimited number of identical copies of themselves (self-renewal) and the ability to give rise to differentiated progeny that can contribute to all tissues of the adult body (pluripotency). Decades of past research have identified many of the genetic determinants of the state of these cells, such as the transcription factors Pou5f1, Sox2 and Nanog. Many other transcription factors and, more recently, epigenetic determinants like histone modifications, have been implicated in the establishment, maintenance and loss of pluripotent stem cell identity. The study of these regulators has been boosted by technological advances in the field of high-throughput sequencing (HTS) that have made it possible to investigate the binding and modification of many proteins on a genome-wide level, resulting in an explosion of the amount of genomic data available to researchers. The challenge is now to effectively use these data and to integrate the manifold measurements into coherent and intelligible models that will actually help to better understand the way in which gene expression in stem cells is regulated to maintain their precarious identity. In this thesis, I first explore the potential of HTS by describing two pilot studies using the technology to investigate global differences in the transcriptional profiles of different cell populations. In both cases, I was able to identify a number of promising candidates that mark and, possibly, explain the phenotypic and functional differences between the cells studied. The pilot studies highlighted a strong requirement for specialised software to deal with the analysis of HTS data. I have developed GeneProf, a powerful computational framework for the integrated analysis of functional genomics experiments. This software platform solves many recurring data analysis challenges and streamlines, simplifies and standardises data analysis work flows promoting transparent and reproducible methodologies. The software offers a graphical, user-friendly interface and integrates expert knowledge to guide researchers through the analysis process. All primary analysis results are supplemented with a range of informative plots and summaries that ease the interpretation of the results. Behind the scenes, computationally demanding tasks are handled remotely on a distributed network of high-performance computers, removing rate-limiting requirements on local hardware set-up. A flexible and modular software design lays the foundations for a scalable and extensible framework that will be expanded to address an even wider range of data analysis tasks in future. Using GeneProf, billions of data points from over a hundred published studies have been re-analysed. The results of these analyses are stored in an web-accessible database as part of the GeneProf system, building up an accessible resource for all life scientists. All results, together with details about the analysis procedures used, can be browsed and examined in detail and all final and intermediate results are available and can instantly be reused and compared with new findings. In an attempt to elucidate the regulatory mechanisms of ESCs, I use this knowledge base to identify high-confidence candidate genes relevant to stem cell characteristics by comparing the transcriptional profiles of ESCs with those of other cell types. Doing so, I describe 229 genes with highly ESC-specific transcription. I then integrate the expression data for these ES-specific genes with genome-wide transcription factor binding and histone modification data. After investigating the global characteristics of these "regulatory inputs", I employ machine learning methods to first cluster subgroups of genes with ESC-specific expression patterns and then to define a "regulatory code" that marks one of the subgroups based on their regulatory signatures. The tightly co-regulated core cluster of genes identified in this analysis contains many known members of the transcriptional circuitry of ESCs and a number of novel candidates that I deem worthy of further investigations thanks to their similarity to their better known counterparts. Integrating these candidates and the regulatory code that drives them into our models of the workings of ESCs might eventually help to refine the ways in which we derive, culture and manipulate these cells - with all its prospective benefits to research and medicine

Functional dissection of the enhancer repertoire in human embryonic stem cells

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation with a massively parallel reporter assay (ChIP-STARR-seq) to identify functional enhancers in human embryonic stem cells (ESCs) genome-wide in a quantitative unbiased manner. Although active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac, and H3K4me1 function as enhancers, with activity markedly changing under naive versus primed culture conditions. We identify an enhancer set associated with functions extending to non-ESC-specific processes. Moreover, although transposable elements associate with putative enhancers, only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalog of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.Barakat et al. use a combination of chromatin immunoprecipitation and a massively parallel reporter assay to identify functional enhancers in primed and naive human embryonic stem cells. This genome-wide catalog of validated enhancers provides a valuable resource for the further dissection of the regulatory genome

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

In mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation

Germline biallelic mutation affecting the transcription factor Helios causes pleiotropic defects of immunity

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (T-regs). Studies in mice have emphasized its role in maintenance of T-reg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T cell subsets remain unresolved. Here, we investigated a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We found that Helios(I325V) retains DNA binding and dimerization properties but loses interaction with several partners, including epigenetic remodelers. Whereas patient T-regs showed increased IL-2 production, patient conventional T cells had decreased accessibility of the IL2 locus and consequently reduced IL-2 production. Reduced chromatin accessibility was not exclusive to the IL2 locus but involved a variety of genes associated with T cell activation. Single-cell RNA sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift toward a proinflammatory, effector-like status in patient CD8(+) T cells. Moreover, patient CD4(+) T cells exhibited a pronounced defect in proliferation with delayed expression of surface checkpoint inhibitors, suggesting an impaired onset of the T cell activation program. Collectively, we identified a previously uncharacterized, germline-encoded inborn error of immunity and uncovered a cell-specific defect in Helios-dependent epigenetic regulation. Binding of Helios with specific partners mediates this regulation, which is ultimately necessary for the transcriptional programs that enable T cell homeostasis in health and disease.Peer reviewe

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application. Keywords: Anti-apoptotic protein inhibitors; Ewing sarcoma; High-content imaging; Phenotypic drug screening; Zebrafish xenograft

Esrrb Is a Direct Nanog Target Gene that Can Substitute for Nanog Function in Pluripotent Cells

SummaryEmbryonic stem cell (ESC) self-renewal efficiency is determined by the level of Nanog expression. However, the mechanisms by which Nanog functions remain unclear, and in particular, direct Nanog target genes are uncharacterized. Here we investigate ESCs expressing different Nanog levels and Nanog−/− cells with distinct functionally inducible Nanog proteins to identify Nanog-responsive genes. Surprisingly, these constitute a minor fraction of genes that Nanog binds. Prominent among Nanog-reponsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding of RNAPolII, and stimulates Esrrb transcription. Overexpression of Esrrb in ESCs maintains cytokine-independent self-renewal and pluripotency. Remarkably, this activity is retained in Nanog−/− ESCs. Moreover, Esrrb can reprogram Nanog−/− EpiSCs and can rescue stalled reprogramming in Nanog−/− pre-iPSCs. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal. These findings are consistent with the functional placement of Esrrb downstream of Nanog

WNT signalling control by KDM5C during development affects cognition

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function

Life-long epigenetic programming of cortical architecture by maternal ‘Western’ diet during pregnancy

Funding: European Research Council (SECRET-CELLS, ERC-2015-AdG-695136; T.H.); Wellcome Trust grant number 094476/Z/10/Z, which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St. Andrews.The evolution of human diets led to preferences toward polyunsaturated fatty acid (PUFA) content with ‘Western’ diets enriched in ω-6 PUFAs. Mounting evidence points to ω-6 PUFA excess limiting metabolic and cognitive processes that define longevity in humans. When chosen during pregnancy, ω-6 PUFA-enriched ‘Western’ diets can reprogram maternal bodily metabolism with maternal nutrient supply precipitating the body-wide imprinting of molecular and cellular adaptations at the level of long-range intercellular signaling networks in the unborn fetus. Even though unfavorable neurological outcomes are amongst the most common complications of intrauterine ω-6 PUFA excess, cellular underpinnings of life-long modifications to brain architecture remain unknown. Here, we show that nutritional ω-6 PUFA-derived endocannabinoids desensitize CB1 cannabinoid receptors, thus inducing epigenetic repression of transcriptional regulatory networks controlling neuronal differentiation. We found that cortical neurons lose their positional identity and axonal selectivity when mouse fetuses are exposed to excess ω-6 PUFAs in utero. Conversion of ω-6 PUFAs into endocannabinoids disrupted the temporal precision of signaling at neuronal CB1 cannabinoid receptors, chiefly deregulating Stat3-dependent transcriptional cascades otherwise required to execute neuronal differentiation programs. Global proteomics identified the immunoglobulin family of cell adhesion molecules (IgCAMs) as direct substrates, with DNA methylation and chromatin accessibility profiling uncovering epigenetic reprogramming at >1400 sites in neurons after prolonged cannabinoid exposure. We found anxiety and depression-like behavioral traits to manifest in adult offspring, which is consistent with genetic models of reduced IgCAM expression, to suggest causality for cortical wiring defects. Overall, our data uncover a regulatory mechanism whose disruption by maternal food choices could limit an offspring’s brain function for life.PostprintPeer reviewe

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal