Publications by authors named "Matthew E Ritchie"

79 Publications

Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development.

Immunity 2021 Apr 13. Epub 2021 Apr 13.

Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia; Single Cell Open Research Endeavour (SCORE), The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia. Electronic address:

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.
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http://dx.doi.org/10.1016/j.immuni.2021.03.012DOI Listing
April 2021

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons.

FASEB J 2021 Mar;35(3):e21320

Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, VIC, Australia.

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.
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http://dx.doi.org/10.1096/fj.202001509RDOI Listing
March 2021

Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.

Nat Cell Biol 2021 03 1;23(3):219-231. Epub 2021 Mar 1.

Immunology Division, Walter and Eliza Hall Institute, Parkville, VIC, Australia.

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative 'early progenitor'-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of 'emergency' haematopoiesis.
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http://dx.doi.org/10.1038/s41556-021-00636-7DOI Listing
March 2021

A guide to creating design matrices for gene expression experiments.

F1000Res 2020 10;9:1444. Epub 2020 Dec 10.

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia.

Differential expression analysis of genomic data types, such as RNA-sequencing experiments, use linear models to determine the size and direction of the changes in gene expression. For RNA-sequencing, there are several established software packages for this purpose accompanied with analysis pipelines that are well described. However, there are two crucial steps in the analysis process that can be a stumbling block for many -- the set up an appropriate model via design matrices and the set up of comparisons of interest via contrast matrices. These steps are particularly troublesome because an extensive catalogue for design and contrast matrices does not currently exist. One would usually search for example case studies across different platforms and mix and match the advice from those sources to suit the dataset they have at hand. This article guides the reader through the basics of how to set up design and contrast matrices. We take a practical approach by providing code and graphical representation of each case study, starting with simpler examples (e.g. models with a single explanatory variable) and move onto more complex ones (e.g. interaction models, mixed effects models, higher order time series and cyclical models). Although our work has been written specifically with a -style pipeline in mind, most of it is also applicable to other software packages for differential expression analysis, and the ideas covered can be adapted to data analysis of other high-throughput technologies. Where appropriate, we explain the interpretation and differences between models to aid readers in their own model choices. Unnecessary jargon and theory is omitted where possible so that our work is accessible to a wide audience of readers, from beginners to those with experience in genomics data analysis.
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http://dx.doi.org/10.12688/f1000research.27893.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873980PMC
December 2020

long-read-tools.org: an interactive catalogue of analysis methods for long-read sequencing data.

Gigascience 2021 Feb;10(2)

Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia.

Background: The data produced by long-read third-generation sequencers have unique characteristics compared to short-read sequencing data, often requiring tailored analysis tools for tasks ranging from quality control to downstream processing. The rapid growth in software that addresses these challenges for different genomics applications is difficult to keep track of, which makes it hard for users to choose the most appropriate tool for their analysis goal and for developers to identify areas of need and existing solutions to benchmark against.

Findings: We describe the implementation of long-read-tools.org, an open-source database that organizes the rapidly expanding collection of long-read data analysis tools and allows its exploration through interactive browsing and filtering. The current database release contains 478 tools across 32 categories. Most tools are developed in Python, and the most frequent analysis tasks include base calling, de novo assembly, error correction, quality checking/filtering, and isoform detection, while long-read single-cell data analysis and transcriptomics are areas with the fewest tools available.

Conclusion: Continued growth in the application of long-read sequencing in genomics research positions the long-read-tools.org database as an essential resource that allows researchers to keep abreast of both established and emerging software to help guide the selection of the most relevant tool for their analysis needs.
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http://dx.doi.org/10.1093/gigascience/giab003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931822PMC
February 2021

Covering all your bases: incorporating intron signal from RNA-seq data.

NAR Genom Bioinform 2020 Sep 22;2(3):lqaa073. Epub 2020 Sep 22.

Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia.

RNA-seq datasets can contain millions of intron reads per library that are typically removed from downstream analysis. Only reads overlapping annotated exons are considered to be informative since mature mRNA is assumed to be the major component sequenced, especially for poly(A) RNA libraries. In this study, we show that intron reads are informative, and through exploratory data analysis of read coverage that intron signal is representative of both pre-mRNAs and intron retention. We demonstrate how intron reads can be utilized in differential expression analysis using our method where a unique set of differentially expressed genes can be detected using intron counts. In exploring read coverage, we also developed the software that quickly and robustly calculates user-defined summary statistics for exonic and intronic regions. Across multiple datasets, enabled us to identify several genes with distinctly retained introns that had similar coverage levels to that of neighbouring exons. The work and ideas presented in this paper is the first of its kind to consider multiple biological sources for intron reads through exploratory data analysis, minimizing bias in discovery and interpretation of results. Our findings open up possibilities for further methods development for intron reads and RNA-seq data in general.
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http://dx.doi.org/10.1093/nargab/lqaa073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671406PMC
September 2020

Single-Cell Transcriptomic Analysis Reveals BCMA CAR-T Cell Dynamics in a Patient with Refractory Primary Plasma Cell Leukemia.

Mol Ther 2021 02 3;29(2):645-657. Epub 2020 Dec 3.

Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China; Institute of Hematology, Zhejiang University, Hangzhou 310058, China; Zhejiang Province Engineering Laboratory for Stem Cell and Immunity Therapy, Hangzhou 310058, China; Zhejiang Laboratory for Systems & Precision Medicine, Zhejiang University Medical Center, Hangzhou 310058, China. Electronic address:

Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 T cells including CAR-T products, CAR-T cells, and endogenous T cells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous T cell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8 CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.
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http://dx.doi.org/10.1016/j.ymthe.2020.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854300PMC
February 2021

is a maternal effect gene required for genomic imprinting.

Elife 2020 11 13;9. Epub 2020 Nov 13.

Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

Genomic imprinting establishes parental allele-biased expression of a suite of mammalian genes based on parent-of-origin specific epigenetic marks. These marks are under the control of maternal effect proteins supplied in the oocyte. Here we report epigenetic repressor as a novel maternal effect gene that regulates the imprinted expression of ten genes in mice. We also found zygotic SMCHD1 had a dose-dependent effect on the imprinted expression of seven genes. Together, zygotic and maternal SMCHD1 regulate three classic imprinted clusters and eight other genes, including non-canonical imprinted genes. Interestingly, the loss of maternal SMCHD1 does not alter germline DNA methylation imprints pre-implantation or later in gestation. Instead, what appears to unite most imprinted genes sensitive to SMCHD1 is their reliance on polycomb-mediated methylation as germline or secondary imprints, therefore we propose that SMCHD1 acts downstream of polycomb imprints to mediate its function.
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http://dx.doi.org/10.7554/eLife.55529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665889PMC
November 2020

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

Nat Immunol 2020 12 19;21(12):1574-1584. Epub 2020 Oct 19.

Immunology Division, The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Linc-KitSca-1Flt3 cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.
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http://dx.doi.org/10.1038/s41590-020-0799-xDOI Listing
December 2020

Therapeutic Reversal of Radiotherapy Injury to Pro-fibrotic Dysfunctional Fibroblasts In Vitro Using Adipose-derived Stem Cells.

Plast Reconstr Surg Glob Open 2020 Mar 25;8(3):e2706. Epub 2020 Mar 25.

Regenerative Surgery and Lymphatics Department, O'Brien Institute, St. Vincent's Institute, Fitzroy, Victoria, Australia.

Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes.

Methods: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFβ) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery.

Results: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFβ was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF.

Conclusions: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.
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http://dx.doi.org/10.1097/GOX.0000000000002706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253248PMC
March 2020

Harnessing Natural Killer Immunity in Metastatic SCLC.

J Thorac Oncol 2020 09 26;15(9):1507-1521. Epub 2020 May 26.

ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia. Electronic address:

Introduction: SCLC is the most aggressive subtype of lung cancer, and though most patients initially respond to platinum-based chemotherapy, resistance develops rapidly. Immunotherapy holds promise in the treatment of lung cancer; however, patients with SCLC exhibit poor overall responses highlighting the necessity for alternative approaches. Natural killer (NK) cells are an alternative to T cell-based immunotherapies that do not require sensitization to antigens presented on the surface of tumor cells.

Methods: We investigated the immunophenotype of human SCLC tumors by both flow cytometry on fresh samples and bioinformatic analysis. Cell lines generated from murine SCLC were transplanted into mice lacking key cytotoxic immune cells. Subcutaneous tumor growth, metastatic dissemination, and activation of CD8 T and NK cells were evaluated by histology and flow cytometry.

Results: Transcriptomic analysis of human SCLC tumors revealed heterogeneous immune checkpoint and cytotoxic signature profiles. Using sophisticated, genetically engineered mouse models, we reported that the absence of NK cells, but not CD8 T cells, substantially enhanced metastatic dissemination of SCLC tumor cells in vivo. Moreover, hyperactivation of NK cell activity through augmentation of interleukin-15 or transforming growth factor-β signaling pathways ameliorated SCLC metastases, an effect that was enhanced when combined with antiprogrammed cell death-1 therapy.

Conclusions: These proof-of-principle findings provide a rationale for exploiting the antitumor functions of NK cells in the treatment of patients with SCLC. Moreover, the distinct immune profiles of SCLC subtypes reveal an unappreciated level of heterogeneity that warrants further investigation in the stratification of patients for immunotherapy.
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http://dx.doi.org/10.1016/j.jtho.2020.05.008DOI Listing
September 2020

Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal.

Nat Commun 2020 05 15;11(1):2420. Epub 2020 May 15.

Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Victoria, 3010, Australia.

Archetypal human pluripotent stem cells (hPSC) are widely considered to be equivalent in developmental status to mouse epiblast stem cells, which correspond to pluripotent cells at a late post-implantation stage of embryogenesis. Heterogeneity within hPSC cultures complicates this interspecies comparison. Here we show that a subpopulation of archetypal hPSC enriched for high self-renewal capacity (ESR) has distinct properties relative to the bulk of the population, including a cell cycle with a very low G1 fraction and a metabolomic profile that reflects a combination of oxidative phosphorylation and glycolysis. ESR cells are pluripotent and capable of differentiation into primordial germ cell-like cells. Global DNA methylation levels in the ESR subpopulation are lower than those in mouse epiblast stem cells. Chromatin accessibility analysis revealed a unique set of open chromatin sites in ESR cells. RNA-seq at the subpopulation and single cell levels shows that, unlike mouse epiblast stem cells, the ESR subset of hPSC displays no lineage priming, and that it can be clearly distinguished from gastrulating and extraembryonic cell populations in the primate embryo. ESR hPSC correspond to an earlier stage of post-implantation development than mouse epiblast stem cells.
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http://dx.doi.org/10.1038/s41467-020-16214-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229198PMC
May 2020

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Blood 2020 08;136(8):957-973

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
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http://dx.doi.org/10.1182/blood.2019002548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441169PMC
August 2020

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Cell Death Differ 2020 Oct 27;27(10):2768-2780. Epub 2020 Apr 27.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.
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http://dx.doi.org/10.1038/s41418-020-0541-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492458PMC
October 2020

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice.

Blood Adv 2020 04;4(7):1270-1283

Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.
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http://dx.doi.org/10.1182/bloodadvances.2019001323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160253PMC
April 2020

Opportunities and challenges in long-read sequencing data analysis.

Genome Biol 2020 02 7;21(1):30. Epub 2020 Feb 7.

Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Australia.

Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.
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http://dx.doi.org/10.1186/s13059-020-1935-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006217PMC
February 2020

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Nat Immunol 2020 Mar;21(3):354

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41590-020-0606-8DOI Listing
March 2020

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Nat Immunol 2020 02 23;21(2):168-177. Epub 2019 Dec 23.

Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.
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http://dx.doi.org/10.1038/s41590-019-0567-yDOI Listing
February 2020

CellBench: R/Bioconductor software for comparing single-cell RNA-seq analysis methods.

Bioinformatics 2020 04;36(7):2288-2290

Epigenetics and Development Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.

Motivation: Bioinformatic analysis of single-cell gene expression data is a rapidly evolving field. Hundreds of bespoke methods have been developed in the past few years to deal with various aspects of single-cell analysis and consensus on the most appropriate methods to use under different settings is still emerging. Benchmarking the many methods is therefore of critical importance and since analysis of single-cell data usually involves multi-step pipelines, effective evaluation of pipelines involving different combinations of methods is required. Current benchmarks of single-cell methods are mostly implemented with ad-hoc code that is often difficult to reproduce or extend, and exhaustive manual coding of many combinations is infeasible in most instances. Therefore, new software is needed to manage pipeline benchmarking.

Results: The CellBench R software facilitates method comparisons in either a task-centric or combinatorial way to allow pipelines of methods to be evaluated in an effective manner. CellBench automatically runs combinations of methods, provides facilities for measuring running time and delivers output in tabular form which is highly compatible with tidyverse R packages for summary and visualization. Our software has enabled comprehensive benchmarking of single-cell RNA-seq normalization, imputation, clustering, trajectory analysis and data integration methods using various performance metrics obtained from data with available ground truth. CellBench is also amenable to benchmarking other bioinformatics analysis tasks.

Availability And Implementation: Available from https://bioconductor.org/packages/CellBench.
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http://dx.doi.org/10.1093/bioinformatics/btz889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141847PMC
April 2020

Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons.

Mol Neurobiol 2020 Feb 19;57(2):949-963. Epub 2019 Oct 19.

Department of Anatomy and Neuroscience, The University of Melbourne, Medical Building (181), Parkville, Victoria, 3010, Australia.

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary processes and elicit a diverse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.
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http://dx.doi.org/10.1007/s12035-019-01782-8DOI Listing
February 2020

Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma.

Nat Commun 2019 09 13;10(1):4190. Epub 2019 Sep 13.

Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The KRAS oncoprotein, a critical driver in 33% of lung adenocarcinoma (LUAD), has remained an elusive clinical target due to its perceived undruggable nature. The identification of dependencies borne through common co-occurring mutations are sought to more effectively target KRAS-mutant lung cancer. Approximately 20% of KRAS-mutant LUAD carry loss-of-function mutations in KEAP1, a negative regulator of the antioxidant response transcription factor NFE2L2/NRF2. We demonstrate that Keap1-deficient Kras lung tumors arise from a bronchiolar cell-of-origin, lacking pro-tumorigenic macrophages observed in tumors originating from alveolar cells. Keap1 loss activates the pentose phosphate pathway, inhibition of which, using 6-AN, abrogated tumor growth. These studies highlight alternative therapeutic approaches to specifically target this unique subset of KRAS-mutant LUAD cancers.
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http://dx.doi.org/10.1038/s41467-019-12164-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744438PMC
September 2019

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Cell Stem Cell 2019 08;25(2):258-272.e9

Australian Centre for Blood Diseases, Monash University, Commercial Road, Melbourne, VIC 3004, Australia. Electronic address:

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.
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http://dx.doi.org/10.1016/j.stem.2019.07.001DOI Listing
August 2019

Benchmarking single cell RNA-sequencing analysis pipelines using mixture control experiments.

Nat Methods 2019 06 27;16(6):479-487. Epub 2019 May 27.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Single cell RNA-sequencing (scRNA-seq) technology has undergone rapid development in recent years, leading to an explosion in the number of tailored data analysis methods. However, the current lack of gold-standard benchmark datasets makes it difficult for researchers to systematically compare the performance of the many methods available. Here, we generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create 'pseudo cells' from up to five distinct cancer cell lines. In total, 14 datasets were generated using both droplet and plate-based scRNA-seq protocols. We compared 3,913 combinations of data analysis methods for tasks ranging from normalization and imputation to clustering, trajectory analysis and data integration. Evaluation revealed pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps.
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http://dx.doi.org/10.1038/s41592-019-0425-8DOI Listing
June 2019

Using long-read sequencing to detect imprinted DNA methylation.

Nucleic Acids Res 2019 05;47(8):e46

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville VIC 3052, Australia.

Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of ∼10, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterize, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions (ICRs) as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new ICRs.
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http://dx.doi.org/10.1093/nar/gkz107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486641PMC
May 2019

Lung morphogenesis is orchestrated through Grainyhead-like 2 (Grhl2) transcriptional programs.

Dev Biol 2018 11 5;443(1):1-9. Epub 2018 Sep 5.

ACRF Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.
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http://dx.doi.org/10.1016/j.ydbio.2018.09.002DOI Listing
November 2018

Smchd1 regulates long-range chromatin interactions on the inactive X chromosome and at Hox clusters.

Nat Struct Mol Biol 2018 09 20;25(9):766-777. Epub 2018 Aug 20.

Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.
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http://dx.doi.org/10.1038/s41594-018-0111-zDOI Listing
September 2018

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

PLoS Comput Biol 2018 08 10;14(8):e1006361. Epub 2018 Aug 10.

Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

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.
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http://dx.doi.org/10.1371/journal.pcbi.1006361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105007PMC
August 2018

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

J Leukoc Biol 2018 07 12;104(1):123-133. Epub 2018 Apr 12.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

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.
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http://dx.doi.org/10.1002/JLB.1MA1217-475RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320667PMC
July 2018

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

Cell Metab 2018 04 8;27(4):935-943.e4. Epub 2018 Mar 8.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1/Pten tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations.
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http://dx.doi.org/10.1016/j.cmet.2018.02.006DOI Listing
April 2018

Easy and efficient ensemble gene set testing with EGSEA.

F1000Res 2017 14;6:2010. Epub 2017 Nov 14.

Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.
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http://dx.doi.org/10.12688/f1000research.12544.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747338PMC
November 2017