Florian Halbritter

Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Summary Embryonic 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.

Specification of Tissue-resident Macrophages During Organogenesis

INTRODUCTION Embryonic development and tissue homeostasis depend on cooperation between specialized cell types. Resident macrophages are professional phagocytes that survey their surroundings; eliminate unfit cells, microorganisms, and metabolic waste; and produce a large range of bioactive molecules and growth factors. Resident macrophages also serve tissue-specific purposes: For example, microglia in the central nervous system support neuronal circuit development, Kupffer cells scavenge blood particles and dying red blood cells in the liver, and alveolar macrophages uptake surfactant and remove airborne pollutants and microbes from the airways. Resident macrophage diversity in adult mice is reflected in tissue-specific gene expression profiles, which may be due to responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types. Altogether, the mechanisms responsible for the generation of tissue-resident macrophage diversity remain unclear. RATIONALE Tissue-resident macrophages originate, at least in part, from mesodermal erythro-myeloid progenitors (EMPs) from the yolk sac, which invade the embryo proper at the onset of organogenesis. These tissue-resident macrophages are also self-maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore hypothesized that resident macrophages represent a founding cell type within most organ anlagen. In this model, the generation of macrophage diversity, as observed in the tissues of postnatal mice, may be integral to organogenesis. RESULTS To test this hypothesis and explore the molecular basis of macrophage diversity in mammals, we performed a spatiotemporal analysis of macrophage development in mice, from embryonic day 9 (E9) to 3 weeks after birth. Unbiased single-cell RNA sequencing (RNA-seq) analysis of CD45+ cells, combined with RNA-seq analyses of sorted cell populations, genetic fate mapping, and in situ analyses, revealed that EMPs give rise to a population of premacrophages (pMacs) that colonize the whole embryo from E9.5, as they acquire a core macrophage differentiation program that includes pattern recognition, scavengers, and cytokine receptors. The chemokine receptor Cx3cr1 is up-regulated in pMacs and is important for embryo colonization, which is delayed in Cx3cr1-deficient embryos. Fate mapping of pMacs using a Tnfrsf11a–Cre reporter labels homogeneously fetal and adult tissue-resident macrophages but not HSCs and their progeny. Transcriptional regulators that identify postnatal tissue-resident macrophages in the brain, liver, kidney, skin, and lung were specifically up-regulated immediately after colonization. These dynamic changes mark the onset of diversification into adult macrophages. We identified Id3 as a Kupffer cell–specific transcriptional regulator. Deletion of Id3 in pMacs resulted in Kupffer cell deficiency but did not affect development of microglia and kidney macrophages. CONCLUSION Our study shows that EMP-derived precursors colonize embryonic tissues and simultaneously acquire a full core macrophage program. This is followed by their diversification into tissue-specific macrophages during organogenesis, likely via the expression of distinct sets of transcriptional regulators. These results indicate that differentiation of tissue-resident macrophages is an integral part of organogenesis and identify a spatiotemporal molecular road map for the generation of macrophage diversity in vivo. Our findings provide a conceptual framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to development, homeostasis, and disease pathogenesis in different tissues and will support efforts to differentiate specialized macrophages in vitro. Specification of tissue-resident macrophages. Erythro-myeloid progenitors (EMPs) from the yolk sac colonize the fetal liver and give rise to macrophage precursors (pMacs) that acquire a core macrophage transcriptional program and colonize the embryo from E9.5 in a Cx3cr1-dependent manner (green arrows). Specification of F4/80+ resident macrophages (brown arrows), starting from E10.25, is initiated by the expression of tissue-specific transcriptional regulators. Id3 (red) is important for Kupffer cell development. Transcription factors noted in blue have been shown to be important for the differentiation or the maintenance of the corresponding macrophage subsets. MΦ, macrophage. Tissue-resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that erythro-myeloid progenitors in mice generate premacrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day 9.5 in a chemokine-receptor–dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis, as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.

Reduced Oct4 Expression Directs a Robust Pluripotent State with Distinct Signaling Activity and Increased Enhancer Occupancy by Oct4 and Nanog

Summary Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4+/− ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation.

Cultured cambial meristematic cells as a source of plant natural products

A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive option for producing many of these compounds. However, it is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To bypass the dedifferentiation step, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Using a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by the morphology of CMCs, their hypersensitivity to γ-irradiation and radiomimetic drugs and their ability to differentiate at high frequency. Suspension culture of CMCs derived from Taxus cuspidata, the source of the key anticancer drug, paclitaxel (Taxol), circumvented obstacles routinely associated with the commercial growth of DDCs. These cells may provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products.

GeneProf: analysis of high-throughput sequencing experiments

Existing tools are hence not sufficient to make highthroughput sequencing data fully accessible to the entire research community. To address these challenges, we developed GeneProf, which combines (i) an easy-to-use data analysis suite that automates the analysis process with (ii) a comprehensive resource of integrated, readily interpretable and reusable analysis results. GeneProf ’s user interface is web-based, obviating the need to install specialized software (Supplementary Figs. 1 and 2). The system uses a dedicated, remote compute cluster to carry out large-scale genomic analyses, simultaneously dealing with many computationally demanding tasks. GeneProf is available online (http://www.geneprof.org/) and is free for academic researchers. Technical details are available in Supplementary Discussion and Supplementary Figure 3. GeneProf simplifies the construction of complex workflows by providing assistive web forms (‘wizards’) that conceal the underlying complexities of workflow programming. These wizards reconceive common, best-practice analysis steps as a series of logical stages, which researchers can customize easily by answering only a few basic questions. Users may change wizard-generated workflows to suit specialized requirements, maintaining full methodological flexibility. GeneProf: analysis of high-throughput sequencing experiments

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.

DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation

Summary Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases.

Selective influence of Sox2 on POU transcription factor binding in embryonic and neural stem cells

Embryonic stem cell (ESC) identity is orchestrated by co‐operativity between the transcription factors (TFs) Sox2 and the class V POU‐TF Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class III POU‐TFs Oct6, Brn1 and Brn2. This raises the question of how Sox2 interacts with POU‐TFs to transcriptionally specify ESCs versus NSCs. Here, we show that Oct4 alone binds the Sox/Oct motif and the octamer‐containing palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In contrast, Oct6 binds preferentially to MORE and is unaffected by Sox2. ChIP‐Seq in NSCs shows the MORE to be the most enriched motif for class III POU‐TFs, including MORE subtypes, and that the Sox/Oct motif is not enriched. These results suggest that in NSCs, co‐operativity between Sox2 and class III POU‐TFs may not occur and that POU‐TF‐driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between Sox2 and POU‐TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into how Oct4 alone can convert NSCs to pluripotency.

GeneProf data: a resource of curated, integrated and reusable high-throughput genomics experiments

GeneProf Data (http://www.geneprof.org) is an open web resource for analysed functional genomics experiments. We have built up a large collection of completely processed RNA-seq and ChIP-seq studies by carefully and transparently reanalysing and annotating high-profile public data sets. GeneProf makes these data instantly accessible in an easily interpretable, searchable and reusable manner and thus opens up the path to the advantages and insights gained from genome-scale experiments to a broader scientific audience. Moreover, GeneProf supports programmatic access to these data via web services to further facilitate the reuse of experimental data across tools and laboratories.

Learning models of relational MDPs using graph kernels

Relational reinforcement learning is the application of reinforcement learning to structured state descriptions. Model-based methods learn a policy based on a known model that comprises a description of the actions and their effects as well as the reward function. If the model is initially unknown, one might learn the model first and then apply the model-based method (indirect reinforcement learning). In this paper, we propose a method for model-learning that is based on a combination of several SVMs using graph kernels. Indeterministic processes can be dealt with by combining the kernel approach with a clustering technique. We demonstrate the validity of the approach by a range of experiments on various Blocksworld scenarios.