The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis.

MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.

Short-term particulate matter contamination severely compromises insect antennal olfactory perception.

The consequences of sub-lethal levels of ambient air pollution are underestimated for insects, for example, the accumulation of particulate matter on sensory receptors located on their antennae may have detrimental effects to their function. Here we show that the density of particulate matter on the antennae of houseflies (Musca domestica) collected from an urban environment increases with the severity of air pollution. A combination of behavioural assays, electroantennograms and transcriptomic analysis provide consistent evidence that a brief exposure to particulate matter pollution compromises olfactory perception of reproductive and food odours in both male and female houseflies. Since particulate matter can be transported thousands of kilometres from its origin, these effects may represent an additional factor responsible for global declines in insect numbers, even in pristine and remote areas.

BAF complex-mediated chromatin relaxation is required for establishment of X chromosome inactivation.

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.

Dissecting the molecular control of Interleukin 6 signaling using the M1 cell line.

Interleukin 6 is the classical member of the IL-6 family of cytokines which triggers activation of the JAK/STAT signaling cascade in cells. IL-6 is a pleiotropic cytokine that acts on many cell types and plays a critical role in immune responses, inflammation, and haematopoiesis. Our understanding of the molecular mechanisms governing IL-6 signaling has been aided by numerous studies of this signal transduction pathway, including those utilising the M1 cell line. Here we discuss the studies that we and others have undertaken using the M1 line to examine IL-6 inducible genes, particularly those targets that acts as negative regulators of signaling. Finally, we present a model for the current understanding of the IL-6 signaling pathway at a structural and mechanistic level.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4ß1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

Chromosomes distribute randomly to, but not within, human neutrophil nuclear lobes.

The proximity pattern and radial distribution of chromosome territories within spherical nuclei are random and non-random, respectively. Whether this distribution pattern is conserved in the partitioned or lobed nuclei of polymorphonuclear cells is unclear. Here we use chromosome paint technology to examine the chromosome territories of all 46 chromosomes in hundreds of single human neutrophils - an abundant and famously polymorphonuclear immune cell. By comparing the distribution of chromosomes to randomly shuffled controls and validating with orthogonal chromosome conformation capture technology, we show for the first time that human chromosomes randomly distribute to neutrophil nuclear lobes, while maintaining a non-random radial distribution within these lobes. Furthermore, we demonstrate that chromosome length correlates with three-dimensional volume not only in neutrophils but other human immune cells. This work demonstrates that chromosomes are largely passive passengers during the neutrophil lobing process but are able to subsequently maintain their macro-level organization within lobes.

Membrane budding is a major mechanism of in vivo platelet biogenesis.

How platelets are produced by megakaryocytes in vivo remains controversial despite more than a century of investigation. Megakaryocytes readily produce proplatelet structures in vitro; however, visualization of platelet release from proplatelets in vivo has remained elusive. We show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet replacement. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. In support of this model, we demonstrate that in mice deficient for NF-E2 (the thrombopoietic master regulator), the absence of membrane budding correlates with failure of in vivo platelet production. Accordingly, we propose that membrane budding, rather than proplatelet formation, supplies the majority of the platelet biomass.

Membrane-associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Rα).

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

Haemopedia RNA-seq: a database of gene expression during haematopoiesis in mice and humans.

During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.

scPipe: A flexible R/Bioconductor preprocessing pipeline for single-cell RNA-sequencing data.

Single-cell RNA sequencing (scRNA-seq) technology allows researchers to profile the transcriptomes of thousands of cells simultaneously. Protocols that incorporate both designed and random barcodes have greatly increased the throughput of scRNA-seq, but give rise to a more complex data structure. There is a need for new tools that can handle the various barcoding strategies used by different protocols and exploit this information for quality assessment at the sample-level and provide effective visualization of these results in preparation for higher-level analyses. To this end, we developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols that include CEL-seq, MARS-seq, Chromium 10X, Drop-seq and Smart-seq. scPipe produces a count matrix that is essential for downstream analysis along with an HTML report that summarises data quality. These results can be used as input for downstream analyses including normalization, visualization and statistical testing. scPipe performs this processing in a few simple R commands, promoting reproducible analysis of single-cell data that is compatible with the emerging suite of open-source scRNA-seq analysis tools available in R/Bioconductor and beyond. The scPipe R package is available for download from https://www.bioconductor.org/packages/scPipe.

PU.1 Is Required for the Developmental Progression of Multipotent Progenitors to Common Lymphoid Progenitors.

The transcription factor PU.1 is required for the development of mature myeloid and lymphoid cells. Due to this essential role and the importance of PU.1 in regulating several signature markers of lymphoid progenitors, its precise function in early lymphopoiesis has been difficult to define. Here, we demonstrate that PU.1 was required for efficient generation of lymphoid-primed multipotent progenitors (LMPPs) from hematopoietic stem cells and was essential for the subsequent formation of common lymphoid progenitors (CLPs). By contrast, further differentiation into the B-cell lineage was independent of PU.1. Examination of the transcriptional changes in conditional progenitors revealed that PU.1 activates lymphoid genes in LMPPs, while repressing genes normally expressed in neutrophils. These data identify PU.1 as a critical regulator of lymphoid priming and the transition between LMPPs and CLPs.

Transcriptional profiling of eosinophil subsets in interleukin-5 transgenic mice.

Eosinophils are important in fighting parasitic infections and are implicated in the pathogenesis of asthma and allergy. IL-5 is a critical regulator of eosinophil development, controlling proliferation, differentiation, and maturation of the lineage. Mice that constitutively express IL-5 have in excess of 10-fold more eosinophils in the hematopoietic organs than their wild type (WT) counterparts. We have identified that much of this expansion is in a population of Siglec-F high eosinophils, which are rare in WT mice. In this study, we assessed transcription in myeloid progenitors, eosinophil precursors, and Siglec-F medium and Siglec-F high eosinophils from IL-5 transgenic mice and in doing so have created a useful resource for eosinophil biologists. We have then utilized these populations to construct an eosinophil trajectory based on gene expression and to identify gene sets that are associated with eosinophil lineage progression. Cell cycle genes were significantly associated with the trajectory, and we experimentally demonstrate an increasing trend toward quiescence along the trajectory. Additionally, we found gene expression changes associated with constitutive IL-5 signaling in eosinophil progenitors, many of which were not observed in eosinophils.

Identification of a Siglec-F+ granulocyte-macrophage progenitor.

In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα- myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα- cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα- population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.

Antennal scales improve signal detection efficiency in moths.

The elaborate bipectinate antennae of male moths are thought to increase their sensitivity to female sex pheromones, and so should be favoured by selection. Yet simple filamentous antennae are the most common structure among moths. The stereotypic arrangements of scales on the surface of antennae may resolve this paradox. We use computational fluid dynamics techniques to model how scales on the filamentous antennae of moths affect the passage of different particles in the airflow across the flagellum in both small and large moths. We found that the scales provide an effective solution to improve the efficacy of filamentous antennae, by increasing the concentration of nanoparticles, which resemble pheromones, around the antennae. The smaller moths have a greater increase in antennal efficiency than larger moths. The scales also divert microparticles, which resemble dust, away from the antennal surface, thereby reducing contamination. The positive correlations between antennal scale angles and sensilla number across Heliozelidae moths are consistent with the predictions of our model.

A preliminary molecular phylogeny of shield-bearer moths (Lepidoptera: Adeloidea: Heliozelidae) highlights rich undescribed diversity.

Heliozelidae are a widespread, evolutionarily early diverging family of small, day-flying monotrysian moths, for which a comprehensive phylogeny is lacking. We generated the first molecular phylogeny of the family using DNA sequences of two mitochondrial genes (COI and COII) and two nuclear genes (H3 and 28S) from 130 Heliozelidae specimens, including eight of the twelve known genera: Antispila, Antispilina, Coptodisca, Heliozela, Holocacista, Hoplophanes, Pseliastis, and Tyriozela. Our results provide strong support for five major Heliozelidae clades: (i) a large widespread clade containing the leaf-mining genera Antispilina, Coptodisca and Holocacista and some species of Antispila, (ii) a clade containing most of the described Antispila, (iii) a clade containing the leaf-mining genus Heliozela and the monotypic genus Tyriozela, (iv) an Australian clade containing Pseliastis and (v) an Australian clade containing Hoplophanes. Each clade includes several new species and potentially new genera. Collectively, our data uncover a rich and undescribed diversity that appears to be especially prevalent in Australia. Our work highlights the need for a major taxonomic revision of the family and for generating a robust molecular phylogeny using multi-gene approaches in order to resolve the relationships among clades.

MiSTIC, an integrated platform for the analysis of heterogeneity in large tumour transcriptome datasets.

Genome-wide transcriptome profiling has enabled non-supervised classification of tumours, revealing different sub-groups characterized by specific gene expression features. However, the biological significance of these subtypes remains for the most part unclear. We describe herein an interactive platform, Minimum Spanning Trees Inferred Clustering (MiSTIC), that integrates the direct visualization and comparison of the gene correlation structure between datasets, the analysis of the molecular causes underlying co-variations in gene expression in cancer samples, and the clinical annotation of tumour sets defined by the combined expression of selected biomarkers. We have used MiSTIC to highlight the roles of specific transcription factors in breast cancer subtype specification, to compare the aspects of tumour heterogeneity targeted by different prognostic signatures, and to highlight biomarker interactions in AML. A version of MiSTIC preloaded with datasets described herein can be accessed through a public web server (http://mistic.iric.ca); in addition, the MiSTIC software package can be obtained (github.com/iric-soft/MiSTIC) for local use with personalized datasets.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

Haemopedia: An Expression Atlas of Murine Hematopoietic Cells.

Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

Setdb1-mediated H3K9 methylation is enriched on the inactive X and plays a role in its epigenetic silencing.

BACKGROUND: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. RESULTS: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. CONCLUSIONS: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.