Bo T. Porse

Bone Marrow-Derived Macrophages (BMM): Isolation and Applications

INTRODUCTIONBone marrow-derived macrophages (BMM) are primary macrophage cells, derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. The efficiency of the differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, the BMMs are suitable for numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages have a unique ability to ingest microbes. We describe a test for the phagocytic efficiency of BMMs by exposing them to fluorescently labeled yeast zymosan bioparticles. Also, a method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is described. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division.

E2F Repression by C/EBPα Is Required for Adipogenesis and Granulopoiesis In Vivo

The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have identified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes

Polycomb group (PcG) proteins are transcriptional repressors, which regulate proliferation and cell fate decisions during development, and their deregulated expression is a frequent event in human tumours. The Polycomb repressive complex 2 (PRC2) catalyzes trimethylation (me3) of histone H3 lysine 27 (K27), and it is believed that this activity mediates transcriptional repression. Despite the recent progress in understanding PcG function, the molecular mechanisms by which the PcG proteins repress transcription, as well as the mechanisms that lead to the activation of PcG target genes are poorly understood. To gain insight into these mechanisms, we have determined the global changes in histone modifications in embryonic stem (ES) cells lacking the PcG protein Suz12 that is essential for PRC2 activity. We show that loss of PRC2 activity results in a global increase in H3K27 acetylation. The methylation to acetylation switch correlates with the transcriptional activation of PcG target genes, both during ES cell differentiation and in MLL-AF9-transduced hematopoietic stem cells. Moreover, we provide evidence that the acetylation of H3K27 is catalyzed by the acetyltransferases p300 and CBP. Based on these data, we propose that the PcG proteins in part repress transcription by preventing the binding of acetyltransferases to PcG target genes.

BLUEPRINT to decode the epigenetic signature written in blood

volume 30 number 3 march 2012 nature biotechnology To the Editor: Last October, scientists gathered in Amsterdam to celebrate the start of BLUEPRINT (http://www.blueprintepigenome.eu/), an EU-funded consortium that will generate epigenomic maps of at least 100 different blood cell types. With this initiative, Europe has pledged a substantial contribution to the ultimate goal of the International Human Epigenome Consortium (IHEC) to map 1,000 human epigenomes. Here, we provide a brief background to the scientific questions that prompted the formation of BLUEPRINT, summarize the overall goals of BLUEPRINT and detail the specific areas in which the consortium will focus its initial efforts and resources. In mammals, nucleated cells share the same genome but have different epigenomes depending on the cell type and many other factors, resulting in an astounding diversity in phenotypic plasticity with respect to morphology and function. This diversity is defined by cell-specific patterns of gene expression, which are controlled through regulatory sites in the genome to which transcription factors bind. In eukaryotes, access to these sites is orchestrated via chromatin, the complex of DNA, RNA and proteins that constitutes the functional platform of the genome. In contrast with DNA, chromatin is not static but highly dynamic, particularly through modifications of histones at nucleosomes and cytosines at the DNA level that together define the epigenome, the epigenetic state of the cell. Advances in new genomics technologies, particularly next-generation sequencing, allow the epigenome to be studied in a holistic fashion, leading to a better understanding of chromatin function and functional annotation of the genome. Yet little is known about how epigenetic characteristics vary between different cell types, in health and disease or among individuals. This lack of a quantitative framework for the dynamics of the epigenome and its determinants is a major hurdle for the translation of epigenetic observations into regulatory models, the identification of associations between epigenotypes and diseases, and the subsequent development of new classes of compounds for disease prevention and treatment. The task, however, is daunting as each of the several hundred cell types in the human body is expected to show specific epigenomic features that are further expected to respond to environmental inputs in time and space. The research community has realized these limitations and the need for concerted action. The IHEC was founded to coordinate large-scale international efforts toward the goal of a comprehensive human epigenome reference atlas (http://www.ihec-epigenomes. org/). The IHEC will coordinate epigenomic mapping and characterization worldwide to avoid redundant research efforts, implement high data quality standards, coordinate data storage, management and analysis, and provide free access to the epigenomes produced. The maps generated under the umbrella of the IHEC contain detailed information on DNA methylation, histone modification, nucleosome occupancy, and corresponding coding and noncoding RNA expression in different normal and diseased cell types. This will allow integration of different layers of epigenetic information for a wide variety of distinct cell types and thus provide a resource for both basic and applied research. BLUEPRINT aims to bridge the gap in our current knowledge between individual components of the epigenome and their functional dynamics through state-of-the-art analysis in a defined set of primarily human hematopoietic cells from healthy and diseased individuals. Mammalian blood formation or hematopoiesis is one of the best-studied systems of stem cell biology. Blood formation can be viewed as a hierarchical process, and classically, differentiation is defined to occur along the myeloid and lymphoid lineages. The identity of cellular intermediates and the geometry of branch points are still under intense investigation and therefore provide a paradigm for delineation of fundamental principles of cell fate determination and regulation of proliferation and lifespan, which differ considerably between different types of blood cells. BLUEPRINT will generate reference epigenomes of at least 50 specific blood cell types and their malignant counterparts and aim to provide high-quality reference epigenomes of primary cells from >60 individuals with detailed genetic and, where appropriate, medical records. To account for and quantify the impact of DNA sequence variation on epigenome differences, BLUEPRINT will work whenever possible on samples of known genetic variation, including samples from the Cambridge BioResource (Cambridge, UK), the International Cancer Genome Consortium and the British Diabetic Twin Study for disease-discordant monozygotic twin samples. The Wellcome Trust Sanger Institute (Hinxton, UK) will also provide full genomic sequencing for up to 100 samples. BLUEPRINT will harness existing proven technologies to generate reference epigenomes, including RNA-Seq for transcriptome analysis, bisulfite sequencing for methylome analysis, DNaseI-Seq for analysis of hypersensitive sites and ChIPSeq for analysis of at least six histone marks. Moreover, BLUEPRINT aims to develop new technologies to enhance high-throughput epigenome mapping, particularly when using few cells. BLUEPRINT is initially focusing on four main areas. One main goal of the project is to comprehensively analyze diverse epigenomic maps and make them available as an integrated BLUEPRINT-IHEC resource to the scientific community. Integration is envisioned for related projects within species (e.g., the 1000 Genomes Project) and between species (e.g., modENCODE) to better understand functional aspects (e.g., shared pathways) and the evolution of cell lineage development. Analysis of the BLUEPRINT data is expected to catalyze a better understanding of the relationship between epigenetic and genomic information and will form the basis for generation of new methods (e.g., epigenetic imputation) for prediction of epigenetic states from epigenomic profiles. Such prediction methods will facilitate a move toward a more quantitative knowledge and modeling of epigenetic mechanisms. As a result, such models could in the future assist in ‘reverse engineering’ of regulatory networks to repair or restore epigenetic codes that have been perturbed by disease. A second goal of BLUEPRINT is to systematically link epigenetic variation with phenotypic plasticity in health and disease. This will be attempted in three ways. First, genetic and epigenetic varation in two blood cell types from 100 healthy individuals will be analyzed. These measurements will be combined with whole-genome and transcriptome sequencing to dissect the interplay between common DNA sequence BLUEPRINT to decode the epigenetic signature written in blood CORRESPONDENCE

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.

microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.

microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.

Regulation of Axon Guidance by Compartmentalized Nonsense-Mediated mRNA Decay

Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we show that Robo3.2, a receptor for the Slit family of guidance cues, is synthesized locally within axons of commissural neurons. Robo3.2 translation is induced by floor-plate-derived signals as axons cross the spinal cord midline. Robo3.2 is also a predicted target of the nonsense-mediated mRNA decay (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding.

Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

CCAAT/enhancer binding protein (C/EBP)α is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which dissociate the ability of C/EBPα to block cell cycle progression through E2F inhibition from its function as a transcriptional activator impair the in vivo development of the neutrophil granulocyte and adipose lineages. We now show that such mutations increase the capacity of bone marrow (BM) myeloid progenitors to proliferate, and predispose mice to a granulocytic myeloproliferative disorder and transformation of the myeloid compartment of the BM. Both of these phenotypes were transplantable into lethally irradiated recipients. BM transformation was characterized by a block in granulocyte differentiation, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations—all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count—normally associated with AML—were absent. These results show that disrupting the cell cycle regulatory function of C/EBPα is sufficient to initiate AML-like transformation of the granulocytic lineage, but only partially the peripheral pathology of AML.

BloodSpot: a database of gene expression profiles and transcriptional programs for healthy and malignant haematopoiesis

Research on human and murine haematopoiesis has resulted in a vast number of gene-expression data sets that can potentially answer questions regarding normal and aberrant blood formation. To researchers and clinicians with limited bioinformatics experience, these data have remained available, yet largely inaccessible. Current databases provide information about gene-expression but fail to answer key questions regarding co-regulation, genetic programs or effect on patient survival. To address these shortcomings, we present BloodSpot (www.bloodspot.eu), which includes and greatly extends our previously released database HemaExplorer, a database of gene expression profiles from FACS sorted healthy and malignant haematopoietic cells. A revised interactive interface simultaneously provides a plot of gene expression along with a Kaplan–Meier analysis and a hierarchical tree depicting the relationship between different cell types in the database. The database now includes 23 high-quality curated data sets relevant to normal and malignant blood formation and, in addition, we have assembled and built a unique integrated data set, BloodPool. Bloodpool contains more than 2000 samples assembled from six independent studies on acute myeloid leukemia. Furthermore, we have devised a robust sample integration procedure that allows for sensitive comparison of user-supplied patient samples in a well-defined haematopoietic cellular space.