School of Mathematics and Statistics - Research Publications

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 14
  • Item
    No Preview Available
    HBO1 (KAT7) Does Not Have an Essential Role in Cell Proliferation, DNA Replication, or Histone 4 Acetylation in Human Cells
    Kueh, AJ ; Eccles, S ; Tang, L ; Garnham, AL ; May, RE ; Herold, MJ ; Smyth, GK ; Voss, AK ; Thomas, T (American Society for Microbiology, 2020-02-01)
    HBO1 (MYST2/KAT7) is essential for histone 3 lysine 14 acetylation (H3K14ac) but is dispensable for H4 acetylation and DNA replication in mouse tissues. In contrast, previous studies using small interfering RNA (siRNA) knockdown in human cell lines have suggested that HBO1 is essential for DNA replication. To determine if HBO1 has distinctly different roles in immortalized human cell lines and normal mouse cells, we performed siRNA knockdown of HBO1. In addition, we used CRISPR/Cas9 to generate 293T, MCF7, and HeLa cell lines lacking HBO1. Using both techniques, we show that HBO1 is essential for all H3K14ac in human cells and is unlikely to have a direct effect on H4 acetylation and only has minor effects on cell proliferation. Surprisingly, the loss of HBO1 and H3K14ac in HeLa cells led to the secondary loss of almost all H4 acetylation after 4 weeks. Thus, HBO1 is dispensable for DNA replication and cell proliferation in immortalized human cells. However, while cell proliferation proceeded without HBO1 and H3K14ac, HBO1 gene deletion led to profound changes in cell adhesion, particularly in 293T cells. Consistent with this phenotype, the loss of HBO1 in both 293T and HeLa principally affected genes mediating cell adhesion, with comparatively minor effects on other cellular processes.
  • Item
    Thumbnail Image
    Activation of stably silenced genes by recruitment of a synthetic de-methylating module
    Chan, WF ; Coughlan, HD ; Chen, Y ; Keenan, CR ; Smyth, GK ; Perkins, AC ; Johanson, TM ; Allan, RS (NATURE PORTFOLIO, 2022-09-23)
    Stably silenced genes that display a high level of CpG dinucleotide methylation are refractory to the current generation of dCas9-based activation systems. To counter this, we create an improved activation system by coupling the catalytic domain of DNA demethylating enzyme TET1 with transcriptional activators (TETact). We show that TETact demethylation-coupled activation is able to induce transcription of suppressed genes, both individually and simultaneously in cells, and has utility across a number of cell types. Furthermore, we show that TETact can effectively reactivate embryonic haemoglobin genes in non-erythroid cells. We anticipate that TETact will expand the existing CRISPR toolbox and be valuable for functional studies, genetic screens and potential therapeutics.
  • Item
    Thumbnail Image
    Loss of TIP60 (KAT5) abolishes H2AZ lysine 7 acetylation and causes p53, INK4A, and ARF-independent cell cycle arrest
    Wichmann, J ; Pitt, C ; Eccles, S ; Garnham, AL ; Li-Wai-Suen, CSN ; May, R ; Allan, E ; Wilcox, S ; Herold, MJ ; Smyth, GK ; Monahan, BJ ; Thomas, T ; Voss, AK (SPRINGERNATURE, 2022-07-20)
    Histone acetylation is essential for initiating and maintaining a permissive chromatin conformation and gene transcription. Dysregulation of histone acetylation can contribute to tumorigenesis and metastasis. Using inducible cre-recombinase and CRISPR/Cas9-mediated deletion, we investigated the roles of the histone lysine acetyltransferase TIP60 (KAT5/HTATIP) in human cells, mouse cells, and mouse embryos. We found that loss of TIP60 caused complete cell growth arrest. In the absence of TIP60, chromosomes failed to align in a metaphase plate during mitosis. In some TIP60 deleted cells, endoreplication occurred instead. In contrast, cell survival was not affected. Remarkably, the cell growth arrest caused by loss of TIP60 was independent of the tumor suppressors p53, INK4A and ARF. TIP60 was found to be essential for the acetylation of H2AZ, specifically at lysine 7. The mRNA levels of 6236 human and 8238 mouse genes, including many metabolism genes, were dependent on TIP60. Among the top 50 differentially expressed genes, over 90% were downregulated in cells lacking TIP60, supporting a role for TIP60 as a key co-activator of transcription. We propose a primary role of TIP60 in H2AZ lysine 7 acetylation and transcriptional activation, and that this fundamental role is essential for cell proliferation. Growth arrest independent of major tumor suppressors suggests TIP60 as a potential anti-cancer drug target.
  • Item
    Thumbnail Image
    Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria
    Studniberg, S ; Ioannidis, LJ ; Utami, RAS ; Trianty, L ; Liao, Y ; Abeysekera, W ; Li-Wai-Suen, CSN ; Pietrzak, HM ; Healer, J ; Puspitasari, AM ; Apriyanti, D ; Coutrier, F ; Poespoprodjo, JR ; Kenangalem, E ; Andries, B ; Prayoga, P ; Sariyanti, N ; Smyth, GK ; Cowman, AF ; Price, RN ; Noviyanti, R ; Shi, W ; Garnham, AL ; Hansen, DS (WILEY, 2022-04)
    Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.
  • Item
    No Preview Available
    Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition
    Hogg, SJ ; Motorna, O ; Cluse, LA ; Johanson, TM ; Coughlan, HD ; Raviram, R ; Myers, RM ; Costacurta, M ; Todorovski, I ; Pijpers, L ; Bjelosevic, S ; Williams, T ; Huskins, SN ; Kearney, CJ ; Devlin, JR ; Fan, Z ; Jabbari, JS ; Martin, BP ; Fareh, M ; Kelly, MJ ; Dupere-Richer, D ; Sandow, JJ ; Feran, B ; Knight, D ; Khong, T ; Spencer, A ; Harrison, SJ ; Gregory, G ; Wickramasinghe, VO ; Webb, A ; Taberlay, PC ; Bromberg, KD ; Lai, A ; Papenfuss, AT ; Smyth, GK ; Allan, RS ; Licht, JD ; Landau, DA ; Abdel-Wahab, O ; Shortt, J ; Vervoort, SJ ; Johnstone, RW (CELL PRESS, 2021-05-20)
    To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
  • Item
    Thumbnail Image
    The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21
    Zotos, D ; Quast, I ; Li-Wai-Suen, CSN ; McKenzie, CI ; Robinson, MJ ; Kan, A ; Smyth, GK ; Hodgkin, PD ; Tarlinton, DM (NATURE PORTFOLIO, 2021-12-09)
    Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.
  • Item
    Thumbnail Image
    R code and downstream analysis objects for the scRNA-seq atlas of normal and tumorigenic human breast tissue
    Chen, Y ; Pal, B ; Lindeman, GJ ; Visvader, JE ; Smyth, GK (NATURE PORTFOLIO, 2022-03-23)
    Breast cancer is a common and highly heterogeneous disease. Understanding cellular diversity in the mammary gland and its surrounding micro-environment across different states can provide insight into cancer development in the human breast. Recently, we published a large-scale single-cell RNA expression atlas of the human breast spanning normal, preneoplastic and tumorigenic states. Single-cell expression profiles of nearly 430,000 cells were obtained from 69 distinct surgical tissue specimens from 55 patients. This article extends the study by providing quality filtering thresholds, downstream processed R data objects, complete cell annotation and R code to reproduce all the analyses. Data quality assessment measures are presented and details are provided for all the bioinformatic analyses that produced results described in the study.
  • Item
    Thumbnail Image
    Calling differentially methylated regions from whole genome bisulphite sequencing with DMRcate
    Peters, TJ ; Buckley, MJ ; Chen, Y ; Smyth, GK ; Goodnow, CC ; Clark, SJ (OXFORD UNIV PRESS, 2021-11-08)
    Whole genome bisulphite sequencing (WGBS) permits the genome-wide study of single molecule methylation patterns. One of the key goals of mammalian cell-type identity studies, in both normal differentiation and disease, is to locate differential methylation patterns across the genome. We discuss the most desirable characteristics for DML (differentially methylated locus) and DMR (differentially methylated region) detection tools in a genome-wide context and choose a set of statistical methods that fully or partially satisfy these considerations to compare for benchmarking. Our data simulation strategy is both biologically informed-employing distribution parameters derived from large-scale consortium datasets-and thorough. We report DML detection ability with respect to coverage, group methylation difference, sample size, variability and covariate size, both marginally and jointly, and exhaustively with respect to parameter combination. We also benchmark these methods on FDR control and computational time. We use this result to backend and introduce an expanded version of DMRcate: an existing DMR detection tool for microarray data that we have extended to now call DMRs from WGBS data. We compare DMRcate to a set of alternative DMR callers using a similarly realistic simulation strategy. We find DMRcate and RADmeth are the best predictors of DMRs, and conclusively find DMRcate the fastest.
  • Item
    Thumbnail Image
    In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis
    Heitink, L ; Whittle, JR ; Vaillant, F ; Capaldo, BD ; Dekkers, JF ; Dawson, CA ; Milevskiy, MJG ; Surgenor, E ; Tsai, M ; Chen, H-R ; Christie, M ; Chen, Y ; Smyth, GK ; Herold, MJ ; Strasser, A ; Lindeman, GJ ; Visvader, JE (WILEY, 2022-03)
    Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.
  • Item
    Thumbnail Image
    Single cell transcriptome atlas of mouse mammary epithelial cells across development
    Pal, B ; Chen, Y ; Milevskiy, MJG ; Vaillant, F ; Prokopuk, L ; Dawson, CA ; Capaldo, BD ; Song, X ; Jackling, F ; Timpson, P ; Lindeman, GJ ; Smyth, GK ; Visvader, JE (BMC, 2021-06-29)
    BACKGROUND: Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. METHODS: The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. RESULTS: The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. CONCLUSIONS: This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.