School of Mathematics and Statistics - Research Publications

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    A non-canonical function of Ezh2 preserves immune homeostasis
    Vasanthakumar, A ; Xu, D ; Lun, ATL ; Kueh, AJ ; van Gisbergen, KPJM ; Iannarella, N ; Li, X ; Yu, L ; Wang, D ; Williams, BRG ; Lee, SCW ; Majewski, IJ ; Godfrey, DI ; Smyth, GK ; Alexander, WS ; Herold, MJ ; Kallies, A ; Nutt, SL ; Allan, RS (WILEY, 2017-04-01)
    Enhancer of zeste 2 (Ezh2) mainly methylates lysine 27 of histone-H3 (H3K27me3) as part of the polycomb repressive complex 2 (PRC2) together with Suz12 and Eed. However, Ezh2 can also modify non-histone substrates, although it is unclear whether this mechanism has a role during development. Here, we present evidence for a chromatin-independent role of Ezh2 during T-cell development and immune homeostasis. T-cell-specific depletion of Ezh2 induces a pronounced expansion of natural killer T (NKT) cells, although Ezh2-deficient T cells maintain normal levels of H3K27me3. In contrast, removal of Suz12 or Eed destabilizes canonical PRC2 function and ablates NKT cell development completely. We further show that Ezh2 directly methylates the NKT cell lineage defining transcription factor PLZF, leading to its ubiquitination and subsequent degradation. Sustained PLZF expression in Ezh2-deficient mice is associated with the expansion of a subset of NKT cells that cause immune perturbation. Taken together, we have identified a chromatin-independent function of Ezh2 that impacts on the development of the immune system.
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    Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids
    Michalak, EM ; Milevskiy, MJG ; Joyce, RM ; Dekkers, JF ; Jamieson, PR ; Pal, B ; Dawson, CA ; Hu, Y ; Orkin, SH ; Alexander, WS ; Lindeman, GJ ; Smyth, GK ; Visvader, JE ; Rawlins, E (PUBLIC LIBRARY SCIENCE, 2018-08-01)
    Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.
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    Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling
    Pal, B ; Chen, Y ; Vaillant, F ; Jamieson, P ; Gordon, L ; Rios, AC ; Wilcox, S ; Fu, N ; Liu, KH ; Jackling, FC ; Davis, MJ ; Lindeman, GJ ; Smyth, GK ; Visvader, JE (NATURE PUBLISHING GROUP, 2017-11-20)
    The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
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    Impaired thermogenesis and adipose tissue development in mice with fat-specific disruption of insulin and IGF-1 signalling
    Boucher, J ; Mori, MA ; Lee, KY ; Smyth, G ; Liew, CW ; Macotela, Y ; Rourk, M ; Bluher, M ; Russell, SJ ; Kahn, CR (NATURE PUBLISHING GROUP, 2012-06-01)
    Insulin and insulin-like growth factor 1 (IGF-1) have important roles in adipocyte differentiation, glucose tolerance and insulin sensitivity. Here to assess how these pathways can compensate for each other, we created mice with a double tissue-specific knockout of insulin and IGF-1 receptors to eliminate all insulin/IGF-1 signalling in fat. These FIGIRKO mice had markedly decreased white and brown fat mass and were completely resistant to high fat diet-induced obesity and age- and high fat diet-induced glucose intolerance. Energy expenditure was increased in FIGIRKO mice despite a >85% reduction in brown fat mass. However, FIGIRKO mice were unable to maintain body temperature when placed at 4 °C. Brown fat activity was markedly decreased in FIGIRKO mice but was responsive to β3-receptor stimulation. Thus, insulin/IGF-1 signalling has a crucial role in the control of brown and white fat development, and, when disrupted, leads to defective thermogenesis and a paradoxical increase in basal metabolic rate.
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    Complementarity and redundancy of IL-22-producing innate lymphoid cells
    Rankin, LC ; Girard-Madoux, MJH ; Seillet, C ; Mielke, LA ; Kerdiles, Y ; Fenis, A ; Wieduwild, E ; Putoczki, T ; Mondot, S ; Lantz, O ; Demon, D ; Papenfuss, AT ; Smyth, GK ; Lamkanfi, M ; Carotta, S ; Renauld, J-C ; Shi, W ; Carpentier, S ; Soos, T ; Arendt, C ; Ugolini, S ; Huntington, ND ; Bez, GT ; Vivier, E (NATURE PUBLISHING GROUP, 2016-02-01)
    Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.
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    Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response
    Tellier, J ; Shi, W ; Minnich, M ; Liao, Y ; Crawford, S ; Smyth, GK ; Kallies, A ; Busslinger, M ; Nutt, SL (NATURE PUBLISHING GROUP, 2016-03-01)
    Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.
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    A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium
    Su, Z ; Labaj, PP ; Li, S ; Thierry-Mieg, J ; Thierry-Mieg, D ; Shi, W ; Wang, C ; Schroth, GP ; Setterquist, RA ; Thompson, JF ; Jones, WD ; Xiao, W ; Xu, W ; Jensen, RV ; Kelly, R ; Xu, J ; Conesa, A ; Furlanello, C ; Gao, H ; Hong, H ; Jafari, N ; Letovsky, S ; Liao, Y ; Lu, F ; Oakeley, EJ ; Peng, Z ; Praul, CA ; Santoyo-Lopez, J ; Scherer, A ; Shi, T ; Smyth, GK ; Staedtler, F ; Sykacek, P ; Tan, X-X ; Thompson, EA ; Vandesompele, J ; Wang, MD ; Wang, J ; Wolfinger, RD ; Zavadil, J ; Auerbach, SS ; Bao, W ; Binder, H ; Blomquist, T ; Brilliant, MH ; Bushel, PR ; Cain, W ; Catalano, JG ; Chang, C-W ; Chen, T ; Chen, G ; Chen, R ; Chierici, M ; Chu, T-M ; Clevert, D-A ; Deng, Y ; Derti, A ; Devanarayan, V ; Dong, Z ; Dopazo, J ; Du, T ; Fang, H ; Fang, Y ; Fasold, M ; Fernandez, A ; Fischer, M ; Furio-Tari, P ; Fuscoe, JC ; Caiment, F ; Gaj, S ; Gandara, J ; Gao, H ; Ge, W ; Gondo, Y ; Gong, B ; Gong, M ; Gong, Z ; Green, B ; Guo, C ; Guo, L ; Guo, L-W ; Hadfield, J ; Hellemans, J ; Hochreiter, S ; Jia, M ; Jian, M ; Johnson, CD ; Kay, S ; Kleinjans, J ; Lababidi, S ; Levy, S ; Li, Q-Z ; Li, L ; Li, L ; Li, P ; Li, Y ; Li, H ; Li, J ; Li, S ; Lin, SM ; Lopez, FJ ; Lu, X ; Luo, H ; Ma, X ; Meehan, J ; Megherbi, DB ; Mei, N ; Mu, B ; Ning, B ; Pandey, A ; Perez-Florido, J ; Perkins, RG ; Peters, R ; Phan, JH ; Pirooznia, M ; Qian, F ; Qing, T ; Rainbow, L ; Rocca-Serra, P ; Sambourg, L ; Sansone, S-A ; Schwartz, S ; Shah, R ; Shen, J ; Smith, TM ; Stegle, O ; Stralis-Pavese, N ; Stupka, E ; Suzuki, Y ; Szkotnicki, LT ; Tinning, M ; Tu, B ; van Deft, J ; Vela-Boza, A ; Venturini, E ; Walker, SJ ; Wan, L ; Wang, W ; Wang, J ; Wang, J ; Wieben, ED ; Willey, JC ; Wu, P-Y ; Xuan, J ; Yang, Y ; Ye, Z ; Yin, Y ; Yu, Y ; Yuan, Y-C ; Zhang, J ; Zhang, KK ; Zhang, W ; Zhang, W ; Zhang, Y ; Zhao, C ; Zheng, Y ; Zhou, Y ; Zumbo, P ; Tong, W ; Kreil, DP ; Mason, CE ; Shi, L (NATURE PUBLISHING GROUP, 2014-09-01)
    We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the US Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed for all examined platforms, including qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.
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    Cell-Type-Specific Transcriptional Profiles of the Dimorphic Pathogen Penicillium marneffei Reflect Distinct Reproductive, Morphological, and Environmental Demands
    Pasricha, S ; Payne, M ; Canovas, D ; Pase, L ; Ngaosuwankul, N ; Beard, S ; Oshlack, A ; Smyth, GK ; Chaiyaroj, SC ; Boyce, KJ ; Andrianopoulos, A (OXFORD UNIV PRESS INC, 2013-11-01)
    Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.
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    Separate-channel analysis of two-channel microarrays: recovering inter-spot information
    Smyth, GK ; Altman, NS (BMC, 2013-05-26)
    BACKGROUND: Two-channel (or two-color) microarrays are cost-effective platforms for comparative analysis of gene expression. They are traditionally analysed in terms of the log-ratios (M-values) of the two channel intensities at each spot, but this analysis does not use all the information available in the separate channel observations. Mixed models have been proposed to analyse intensities from the two channels as separate observations, but such models can be complex to use and the gain in efficiency over the log-ratio analysis is difficult to quantify. Mixed models yield test statistics for the null distributions can be specified only approximately, and some approaches do not borrow strength between genes. RESULTS: This article reformulates the mixed model to clarify the relationship with the traditional log-ratio analysis, to facilitate information borrowing between genes, and to obtain an exact distributional theory for the resulting test statistics. The mixed model is transformed to operate on the M-values and A-values (average log-expression for each spot) instead of on the log-expression values. The log-ratio analysis is shown to ignore information contained in the A-values. The relative efficiency of the log-ratio analysis is shown to depend on the size of the intraspot correlation. A new separate channel analysis method is proposed that assumes a constant intra-spot correlation coefficient across all genes. This approach permits the mixed model to be transformed into an ordinary linear model, allowing the data analysis to use a well-understood empirical Bayes analysis pipeline for linear modeling of microarray data. This yields statistically powerful test statistics that have an exact distributional theory. The log-ratio, mixed model and common correlation methods are compared using three case studies. The results show that separate channel analyses that borrow strength between genes are more powerful than log-ratio analyses. The common correlation analysis is the most powerful of all. CONCLUSIONS: The common correlation method proposed in this article for separate-channel analysis of two-channel microarray data is no more difficult to apply in practice than the traditional log-ratio analysis. It provides an intuitive and powerful means to conduct analyses and make comparisons that might otherwise not be possible.
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    HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses
    Bolden, JE ; Shi, W ; Jankowski, K ; Kan, C-Y ; Cluse, L ; Martin, BP ; MacKenzie, KL ; Smyth, GK ; Johnstone, RW (NATURE PUBLISHING GROUP, 2013-02-01)
    The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.