During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells. We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variability.

Pluripotency is accompanied by the erasure of parental epigenetic memory, with naïve pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, co-expression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modeling, we show that this variability is associated with coherent, genome-scale oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.

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 Smchd1 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.

While several resources exist that interpret therapeutic significance of genomic alterations in cancer, many regional real-world issues limit access to drugs. There is a need for a pragmatic, evidence-based, context-adapted tool to guide clinical management based on molecular biomarkers. To this end, we have structured a compendium of approved and experimental therapies with associated biomarkers following a survey of drug regulatory databases, existing knowledge bases, and published literature. Each biomarker-disease-therapy triplet was categorised using a tiering system reflective of key therapeutic considerations: approved and reimbursed therapies with respect to a jurisdiction (Tier 1), evidence of efficacy or approval in another jurisdiction (Tier 2), evidence of antitumour activity (Tier 3), and plausible biological rationale (Tier 4). Two resistance categories were defined: lack of efficacy (Tier R1) or antitumor activity (Tier R2). Based on this framework, we curated a digital resource focused on drugs relevant in the Australian healthcare system (TOPOGRAPH: Therapy Oriented Precision Oncology Guidelines for Recommending Anticancer Pharmaceuticals). As of November 2020, TOPOGRAPH comprised 2810 biomarker-disease-therapy triplets in 989 expert-appraised entries, including 373 therapies, 199 biomarkers, and 106 cancer types. In the 345 therapies catalogued, 84 (24%) and 65 (19%) were designated Tiers 1 and 2, respectively, while 271 (79%) therapies were supported by preclinical studies, early clinical trials, retrospective studies, or case series (Tiers 3 and 4). A companion algorithm was also developed to support rational, context-appropriate treatment selection informed by molecular biomarkers. This framework can be readily adapted to build similar resources in other jurisdictions to support therapeutic decision-making.

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy's Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy's algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy's Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm .

We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.

Mammary gland development is coupled to reproductive events by hormonal cues of ovarian and pituitary origin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. In this study KIBRA was identified as a transcript showing decreased expression associated with failed mammary gland development in Prlr knockout mammary epithelium. It is strongly up-regulated during pregnancy, falls during lactation and is again up-regulated during involution of the gland at weaning. A bioinformatic approach was undertaken to identify potential binding partners which interact with the WW domains of KIBRA. We show that KIBRA binds to a WW domain binding motif, PPxY, in the tyrosine kinase receptor DDR1, and dissociates upon treatment with the DDR1 ligands collagen type I or IV. In addition we show that KIBRA and DDR1 also interact with PKCz to form a trimeric complex. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodeling events of mammary gland development.

Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful.

Global changes in DNA methylation are observed in development and disease, and single-cell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of average DNA methylation levels. By targeting high-copy SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each scTEM-seq library. Parallel transcriptome analysis is also performed to link global DNA methylation estimates with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Our method reveals global DNA methylation heterogeneity induced by decitabine treatment of KG1a cells associated with altered expression of immune process genes. We also compare global DNA methylation estimates to expression of transposable elements and find a predominance of negative correlations. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond.

Embryonic development is dependent on the maternal supply of proteins through the oocyte, including factors setting up the adequate epigenetic patterning of the zygotic genome. We previously reported that one such factor is the epigenetic repressor SMCHD1, whose maternal supply controls autosomal imprinted expression in mouse preimplantation embryos and mid-gestation placenta. In mouse preimplantation embryos, X chromosome inactivation is also an imprinted process. Combining genomics and imaging, we show that maternal SMCHD1 is required not only for the imprinted expression of Xist in preimplantation embryos, but also for the efficient silencing of the inactive X in both the preimplantation embryo and mid-gestation placenta. These results expand the role of SMCHD1 in enforcing the silencing of Polycomb targets. The inability of zygotic SMCHD1 to fully restore imprinted X inactivation further points to maternal SMCHD1's role in setting up the appropriate chromatin environment during preimplantation development, a critical window of epigenetic remodelling.

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

No abstract available