Publications by authors named "Heather J Lee"

32 Publications

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

Unravelling the Epigenome of Myelodysplastic Syndrome: Diagnosis, Prognosis, and Response to Therapy.

Cancers (Basel) 2020 Oct 26;12(11). Epub 2020 Oct 26.

Department of Haematology, Calvary Mater Newcastle, Waratah, NSW 2298, Australia.

Myelodysplastic syndrome (MDS) is a malignancy that disrupts normal blood cell production and commonly affects our ageing population. MDS patients are diagnosed using an invasive bone marrow biopsy and high-risk MDS patients are treated with hypomethylating agents (HMAs) such as decitabine and azacytidine. However, these therapies are only effective in 50% of patients, and many develop resistance to therapy, often resulting in bone marrow failure or leukemic transformation. Therefore, there is a strong need for less invasive, diagnostic tests for MDS, novel markers that can predict response to therapy and/or patient prognosis to aid treatment stratification, as well as new and effective therapeutics to enhance patient quality of life and survival. Epigenetic modifiers such as DNA methylation, long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs) are perturbed in MDS blasts and the bone marrow micro-environment, influencing disease progression and response to therapy. This review focusses on the potential utility of epigenetic modifiers in aiding diagnosis, prognosis, and predicting treatment response in MDS, and touches on the need for extensive and collaborative research using single-cell technologies and multi-omics to test the clinical utility of epigenetic markers for MDS patients in the future.
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http://dx.doi.org/10.3390/cancers12113128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692163PMC
October 2020

Single-cell epigenomics in cancer: charting a course to clinical impact.

Epigenomics 2020 07 13;12(13):1139-1151. Epub 2020 Aug 13.

School of Biomedical Sciences & Pharmacy, Faculty of Health & Medicine, University of Newcastle, Callaghan 2308, New South Wales, Australia.

Cancer is a disease of global epigenetic dysregulation. Mutations in epigenetic regulators are common events in multiple cancer types and epigenetic therapies are emerging as a treatment option in several malignancies. A major challenge for the clinical management of cancer is the heterogeneous nature of this disease. Cancers are composed of numerous cell types and evolve over time. This heterogeneity confounds decisions regarding treatment and promotes disease relapse. The emergence of single-cell epigenomic technologies has introduced the exciting possibility of linking genetic and transcriptional heterogeneity in the context of cancer biology. The next challenge is to leverage these tools for improved patient outcomes. Here we consider how single-cell epigenomic technologies may address the current challenges faced by cancer clinicians.
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http://dx.doi.org/10.2217/epi-2020-0046DOI Listing
July 2020

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia.

Cancer Cell 2019 12;36(6):660-673.e11

Department of Pediatric Oncology, Dana-Farber Cancer Institute, and Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02215, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ccell.2019.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227117PMC
December 2019

Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency.

Cell Syst 2018 07;7(1):63-76.e12

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK; Epigenetics Programme, Babraham Institute, Cambridge, UK; Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cels.2018.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066359PMC
July 2018

scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells.

Nat Commun 2018 02 22;9(1):781. Epub 2018 Feb 22.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK.

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.
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http://dx.doi.org/10.1038/s41467-018-03149-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823944PMC
February 2018

Genome-Wide Analysis of DNA Methylation in Single Cells Using a Post-bisulfite Adapter Tagging Approach.

Methods Mol Biol 2018 ;1712:87-95

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058, Basel, Switzerland.

DNA methylation is an epigenetic mark implicated in the regulation of key biological processes. Using high-throughput sequencing technologies and bisulfite-based approaches, it is possible to obtain comprehensive genome-wide maps of the mammalian DNA methylation landscape with a single-nucleotide resolution and absolute quantification. However, these methods were only applicable to bulk populations of cells. Here, we present a protocol to perform whole-genome bisulfite sequencing on single cells (scBS-Seq) using a post-bisulfite adapter tagging approach. In this method, bisulfite treatment is performed prior to library generation in order to both convert unmethylated cytosines and fragment DNA to an appropriate size. Then DNA fragments are pre-amplified with concomitant integration of the sequencing adapters, and libraries are subsequently amplified and indexed by PCR. Using scBS-Seq we can accurately measure DNA methylation at up to 50% of individual CpG sites and 70% of CpG islands.
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http://dx.doi.org/10.1007/978-1-4939-7514-3_7DOI Listing
July 2018

Erratum to: DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning.

Genome Biol 2017 05 12;18(1):90. Epub 2017 May 12.

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.

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http://dx.doi.org/10.1186/s13059-017-1233-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429579PMC
May 2017

DeepCpG: accurate prediction of single-cell DNA methylation states using deep learning.

Genome Biol 2017 Apr 11;18(1):67. Epub 2017 Apr 11.

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.

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.
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http://dx.doi.org/10.1186/s13059-017-1189-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387360PMC
April 2017

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

Nat Protoc 2017 03 9;12(3):534-547. Epub 2017 Feb 9.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
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http://dx.doi.org/10.1038/nprot.2016.187DOI Listing
March 2017

Tracking the embryonic stem cell transition from ground state pluripotency.

Development 2017 04 7;144(7):1221-1234. Epub 2017 Feb 7.

Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Cambridge CB2 1QR, UK

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
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http://dx.doi.org/10.1242/dev.142711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399622PMC
April 2017

Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

Genome Biol 2016 Apr 18;17:72. Epub 2016 Apr 18.

Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT, UK.

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.
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http://dx.doi.org/10.1186/s13059-016-0944-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834828PMC
April 2016

Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.

Nat Methods 2016 Mar 11;13(3):229-232. Epub 2016 Jan 11.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

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.
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http://dx.doi.org/10.1038/nmeth.3728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770512PMC
March 2016

ELF5 isoform expression is tissue-specific and significantly altered in cancer.

Breast Cancer Res 2016 Jan 7;18(1). Epub 2016 Jan 7.

Cancer Division, Garvan Institute of Medical Research/The Kinghorn Cancer Centre, Sydney, NSW, 2010, Australia.

Background: E74-like factor 5 (ELF5) is an epithelial-specific member of the E26 transforming sequence (ETS) transcription factor family and a critical regulator of cell fate in the placenta, pulmonary bronchi, and milk-producing alveoli of the mammary gland. ELF5 also plays key roles in malignancy, particularly in basal-like and endocrine-resistant forms of breast cancer. Almost all genes undergo alternative transcription or splicing, which increases the diversity of protein structure and function. Although ELF5 has multiple isoforms, this has not been considered in previous studies of ELF5 function.

Methods: RNA-sequencing data for 6757 samples from The Cancer Genome Atlas were analyzed to characterize ELF5 isoform expression in multiple normal tissues and cancers. Extensive in vitro analysis of ELF5 isoforms, including a 116-gene quantitative polymerase chain reaction panel, was performed in breast cancer cell lines.

Results: ELF5 isoform expression was found to be tissue-specific due to alternative promoter use but altered in multiple cancer types. The normal breast expressed one main isoform, while in breast cancer there were subtype-specific alterations in expression. Expression of other ETS factors was also significantly altered in breast cancer, with the basal-like subtype demonstrating a distinct ETS expression profile. In vitro inducible expression of the full-length isoforms 1 and 2, as well as isoform 3 (lacking the Pointed domain) had similar phenotypic and transcriptional effects.

Conclusions: Alternative promoter use, conferring differential regulatory responses, is the main mechanism governing ELF5 action rather than differential transcriptional activity of the isoforms. This understanding of expression and function at the isoform level is a vital first step in realizing the potential of transcription factors such as ELF5 as prognostic markers or therapeutic targets in cancer.
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http://dx.doi.org/10.1186/s13058-015-0666-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704400PMC
January 2016

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

PLoS Biol 2015 Dec 30;13(12):e1002330. Epub 2015 Dec 30.

Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Darlinghurst, New South Wales, Australia.

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.
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http://dx.doi.org/10.1371/journal.pbio.1002330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696735PMC
December 2015

Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.

Nat Methods 2014 Aug 20;11(8):817-820. Epub 2014 Jul 20.

Epigenetics Programme, Babraham Institute, Cambridge, UK.

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.
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http://dx.doi.org/10.1038/nmeth.3035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117646PMC
August 2014

Reprogramming the methylome: erasing memory and creating diversity.

Cell Stem Cell 2014 Jun;14(6):710-9

Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK. Electronic address:

The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian development. Epigenetic reprogramming results in global hypomethylation of the genome together with a profound loss of memory, which underlies naive pluripotency. Such global reprogramming occurs in primordial germ cells, early embryos, and embryonic stem cells where reciprocal molecular links connect the methylation machinery to pluripotency. Priming for differentiation is initiated upon exit from pluripotency, and we propose that epigenetic mechanisms create diversity of transcriptional states, which help with symmetry breaking during cell fate decisions and lineage commitment.
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http://dx.doi.org/10.1016/j.stem.2014.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051243PMC
June 2014

Etoposide induces nuclear re-localisation of AID.

PLoS One 2013 4;8(12):e82110. Epub 2013 Dec 4.

Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082110PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3852760PMC
September 2014

FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.

Cell Stem Cell 2013 Sep 11;13(3):351-9. Epub 2013 Jul 11.

Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK.

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.
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http://dx.doi.org/10.1016/j.stem.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765959PMC
September 2013

Progesterone drives mammary secretory differentiation via RankL-mediated induction of Elf5 in luminal progenitor cells.

Development 2013 Apr 5;140(7):1397-401. Epub 2013 Mar 5.

Cancer Research Program, Garvan Institute of Medical Research, 348 Victoria Street, and St Vincent's Hospital Clinical School, University of New South Wales, Darlinghurst NSW 2010, Australia.

Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epithelial progenitor cells. We demonstrate that Elf5 expression is induced by progesterone and that Elf5 and progesterone cooperate to promote alveolar development. The progesterone receptor and Elf5 are expressed in a mutually exclusive pattern, and we identify RankL as the paracrine mediator of the effects of progesterone on Elf5 expression in CD61+ progenitor cells and their consequent differentiation. Blockade of RankL action prevented progesterone-induced side branching and the expansion of Elf5(+) mature luminal cells. These findings describe a mechanism by which steroid hormones can produce the expansion of steroid hormone receptor-negative mammary epithelial cells.
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http://dx.doi.org/10.1242/dev.088948DOI Listing
April 2013

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

PLoS Biol 2012 27;10(12):e1001461. Epub 2012 Dec 27.

Cancer Research Program and The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

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.
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http://dx.doi.org/10.1371/journal.pbio.1001461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531499PMC
May 2013

Elf5, hormones and cell fate.

Trends Endocrinol Metab 2012 Jun 30;23(6):292-8. Epub 2012 Mar 30.

Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia.

Recent elucidation of the stem and progenitor cell hierarchies that operate during normal tissue and organ development has provided a foundation for the development of new insights into the disease process. These hierarchies are established by genetic mechanisms, which specify and determine cell fate and act as cell-clade gatekeepers, upon which all multicellular organisms depend for viability. Perturbation of this gatekeeper function characterizes developmentally based diseases, such as cancer. Here, the emerging gatekeeper and master regulatory roles of the ETS transcription factor Elf5 in several diverse developmental scenarios is reviewed, and how this function intersects with hormonal and growth factor mediated regulation of these processes is shown.
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http://dx.doi.org/10.1016/j.tem.2012.02.006DOI Listing
June 2012

Interplay between progesterone and prolactin in mammary development and implications for breast cancer.

Mol Cell Endocrinol 2012 Jun 16;357(1-2):101-7. Epub 2011 Sep 16.

Cancer Research Program, Garvan Institute, Sydney, NSW, Australia.

Progesterone and prolactin remodel mammary morphology during pregnancy by acting on the mammary epithelial cell hierarchy. The roles of each hormone in mammary development have been well studied, but evidence of signalling cross-talk between progesterone and prolactin is still emerging. Factors such as receptor activator of NFkB ligand (RANKL) may integrate signals from both hormones to orchestrate their joint actions on the epithelial cell hierarchy. Common targets of progesterone and prolactin signalling are also likely to integrate their pro-proliferative actions in breast cancer. Therefore, a thorough understanding of the interplay between progesterone and prolactin in mammary development may reveal therapeutic targets for breast cancer. This review summarises our understanding of Pg and PRL action in mammary gland development before focusing on molecular mechanisms of signalling cross-talk and the implications for breast cancer.
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http://dx.doi.org/10.1016/j.mce.2011.09.020DOI Listing
June 2012

Lineage specific methylation of the Elf5 promoter in mammary epithelial cells.

Stem Cells 2011 Oct;29(10):1611-9

Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia.

Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E-twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the Elf5 promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether Elf5 promoter methylation plays a role in lineage commitment during mammary development. An increase in Elf5 expression was associated with decreasing Elf5 promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased Elf5 expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the Elf5 promoter is methylated and silenced in the basal, stem cell-containing population relative to luminal cells. These results demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages.
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http://dx.doi.org/10.1002/stem.706DOI Listing
October 2011

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer.

Nature 2010 Nov 29;468(7320):98-102. Epub 2010 Sep 29.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030 Vienna, Austria.

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f(hi) stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.
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http://dx.doi.org/10.1038/nature09387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084017PMC
November 2010

Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants.

Funct Integr Genomics 2010 Mar 15;10(1):87-95. Epub 2009 Oct 15.

Department of Zoology, University of Melbourne, VIC, Australia.

Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes.
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http://dx.doi.org/10.1007/s10142-009-0140-0DOI Listing
March 2010

Mudskippers brood their eggs in air but submerge them for hatching.

J Exp Biol 2007 Nov;210(Pt 22):3946-54

Institute for East China Sea Research, Nagasaki University, Tairamachi, Nagasaki 851-2213, Japan.

Intertidal mudflats are highly productive ecosystems that impose severe environmental challenges on their occupants due to tidal oscillations and extreme shifts in habitat conditions. Reproduction on mudflats requires protection of developing eggs from thermal and salinity extremes, O(2) shortage, dislodgement by currents, siltation and predation. Mudskippers are air-breathing, amphibious fishes, and one of few vertebrates that reside on mudflats. They lay their eggs in mud burrows containing extremely hypoxic water, raising the question of how the eggs survive. We found that the Japanese mudskipper Periophthalmus modestus deposits its eggs on the walls of an air-filled chamber within its burrow. To ensure adequate O(2) for egg development, the burrow-guarding male mudskipper deposits mouthfuls of fresh air into the egg chamber during each low tide, a behaviour that can be upregulated by egg-chamber hypoxia. When egg development is complete the male, on a nocturnal rising tide, removes the egg-chamber air and releases it outside the burrow. This floods the egg chamber and induces egg hatching. Thus, P. modestus has developed a reproductive strategy that allows it to nurture eggs in this severe habitat rather than migrating away from the mudflat. This requires that mudskipper eggs be specialized to develop in air and that the air-breathing capacity of the egg-guarding male be integrated in a complex behavioural repertoire that includes egg guarding, ferrying air to and from the egg chamber, and sensing O(2) levels therein, all in concert with the tidal cycle.
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http://dx.doi.org/10.1242/jeb.010686DOI Listing
November 2007

Effect of hamstring-emphasized resistance training on hamstring:quadriceps strength ratios.

J Strength Cond Res 2007 Feb;21(1):41-7

Sports Injury Research Center, Department of Kinesiology, University of Nevada, Las Vegas 89154, USA.

A decreased hamstring:quadriceps (H:Q) ratio may put the hamstrings and anterior cruciate ligament (ACL) at increased risk of injury. Therefore, the purpose of this study was to evaluate H:Q ratios of 12 female National Collegiate Athletic Association soccer players, and to test the effects of a 6-week strength training program on these ratios. Each subject completed 2 practice sessions before a pretest. Subjects then completed 6 weeks of strength training that included the addition of 2 hamstring specific exercises, followed by a posttest. Peak torque during concentric and eccentric actions for both hamstrings and quadriceps was measured with an isokinetic dynamometer. Each muscle action was tested at 3 angular velocities in the following order: concentric 240, 180, and 60 degrees x s(-1) and eccentric 60, 180, and 240 degrees x s(-1). The H:Q strength ratio was evaluated using concentric muscle actions (concentric hamstrings:concentric quadriceps). This method is commonly used and is thus called the conventional ratio. Because concentric actions do not occur simultaneously in opposing muscles, a more functional assessment compares eccentric hamstring actions to concentric quadriceps actions. This functional ratio was also analyzed. Mean conventional and functional H:Q ratio data were analyzed using separate analysis of variance procedures with repeated measures on all factors (2 [Test] x 2 [Leg] x 3 [Angular Velocity]). The results revealed a significant main effect for factor (F test) with the functional ratio (p < 0.05) but not for the conventional ratio. The mean functional ratio increased from 0.96 +/- 0.09 in pretest to 1.08 +/- 0.11 in posttest. These results suggest that 6 weeks of strength training that emphasizes hamstrings is sufficient to significantly increase the functional ratio. The functional ratio after training exceeded 1.0, which is specifically recommended for prevention of ACL injuries.
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http://dx.doi.org/10.1519/R-18795.1DOI Listing
February 2007

Regulation of growth hormone signaling by selective estrogen receptor modulators occurs through suppression of protein tyrosine phosphatases.

Endocrinology 2007 May 1;148(5):2417-23. Epub 2007 Feb 1.

Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.

Activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway by GH is terminated by the suppressors of cytokine signaling (SOCSs) and protein tyrosine phosphatases, Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Based on our recent report that estrogen inhibits GH signaling by stimulating SOCS-2 expression, we investigated the effects of selective estrogen receptor modulators (SERMs) on GH signaling in human embryonic kidney (HEK293) and breast cancer (MDA-MB-231) cells expressing human GH receptor and estrogen receptor-alpha. 17beta-estradiol (E(2)) suppressed GH activation of a STAT5-responsive luciferase reporter and JAK2 phosphorylation in both cell models. 4-hydroxytamoxifen and raloxifene augmented these actions of GH in HEK293 cells but not breast cancer cells. SOCS-2 expression in both cell types was stimulated by E(2) but unaffected by SERMs. In HEK293 cells, SHP-1 was inhibited by raloxifene and 4-hydroxytamoxifen, whereas the latter additionally inhibited SHP-2. The phosphatases were unaffected by E(2). In breast cancer cells, phosphatase activity was not altered by SERMs or E(2). In summary, estrogen inhibited the JAK2/STAT5 signaling of GH and stimulated SOCS-2 expression in both HEK293 and breast cancer cells. By contrast, SERMs augmented GH signaling by reducing SHP activities in HEK293 cells and had no effect on both in breast cancer cells. We provide the first evidence for a novel mechanism regulating GH signaling, in which SERMs enhance GH activation of the JAK2/STAT5 pathway in a cell-type-dependent manner by attenuating protein tyrosine phosphatase activities.
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http://dx.doi.org/10.1210/en.2006-1305DOI Listing
May 2007

Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation.

Biochem J 2007 May;404(1):141-9

School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.

The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.
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http://dx.doi.org/10.1042/BJ20061826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1868832PMC
May 2007