Publications by authors named "Michelle C Barton"

37 Publications

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

Cell Rep 2021 Nov;37(8):110044

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5 stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.
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http://dx.doi.org/10.1016/j.celrep.2021.110044DOI Listing
November 2021

Oncogenic Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis.

Cancer Discov 2021 Aug 10;11(8):2094-2111. Epub 2021 Apr 10.

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the tumor suppressor in nearly 70% of patients. Most alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between effectors and mutant p53 remains largely undefined. We show that oncogenic effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor , activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic and mutant p53 to mitigate metastasis. SIGNIFICANCE: Oncogenic and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8338884PMC
August 2021

CD8 T cells inhibit metastasis and CXCL4 regulates its function.

Br J Cancer 2021 07 1;125(2):176-189. Epub 2021 Apr 1.

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival.

Methods: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well.

Results: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8 T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8 T-cell function. TCGA pan-cancer data confirmed that CD8Platelet patients have a significantly lower survival probability compared to CD8Platelet.

Conclusions: CD8 T cells inhibit metastasis. When the balance between CD8 T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8 T-cell function.
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http://dx.doi.org/10.1038/s41416-021-01338-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292398PMC
July 2021

Mesoscopic protein-rich clusters host the nucleation of mutant p53 amyloid fibrils.

Proc Natl Acad Sci U S A 2021 03;118(10)

William A. Brookshire Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204;

The protein p53 is a crucial tumor suppressor, often called "the guardian of the genome"; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.
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http://dx.doi.org/10.1073/pnas.2015618118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958401PMC
March 2021

Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo.

Sci Adv 2020 Aug 5;6(32):eaba8415. Epub 2020 Aug 5.

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in , which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional allele in mice. We demonstrate that loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions ( loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.
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http://dx.doi.org/10.1126/sciadv.aba8415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406367PMC
August 2020

KRAB domain of ZFP568 disrupts TRIM28-mediated abnormal interactions in cancer cells.

NAR Cancer 2020 Jun 18;2(2):zcaa007. Epub 2020 May 18.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Interactions of KRAB (Krüppel-associated box)-associated protein KAP1 [also known as TRIM28 (tripartite motif containing protein 28)] with DNA-binding KRAB zinc finger (KRAB-ZF) proteins silence many transposable elements during embryogenesis. However, in some cancers, TRIM28 is upregulated and interacts with different partners, many of which are transcription regulators such as EZH2 in MCF7 cells, to form abnormal repressive or activating complexes that lead to misregulation of genes. We ask whether a KRAB domain-the TRIM28 interaction domain present in native binding partners of TRIM28 that mediate repression of transposable elements-could be used as a tool molecule to disrupt aberrant TRIM28 complexes. Expression of KRAB domain containing fragments from a KRAB-ZF protein (ZFP568) in MCF7 cells, without the DNA-binding zinc fingers, inhibited TRIM28-EZH2 interactions and caused degradation of both TRIM28 and EZH2 proteins as well as other components of the EZH2-associated polycomb repressor 2 complex. In consequence, the product of EZH2 enzymatic activity, trimethylation of histone H3 lysine 27 level, was significantly reduced. The expression of a synthetic KRAB domain significantly inhibits the growth of breast cancer cells (MCF7) but has no effect on normal (immortalized) human mammary epithelial cells (MCF10a). Further, we found that TRIM28 is a positive regulator of TRIM24 protein levels, as observed previously in prostate cancer cells, and expression of the KRAB domain also lowered TRIM24 protein. Importantly, reduction of TRIM24 levels, by treatment with either the KRAB domain or a small-molecule degrader targeted to TRIM24, is accompanied by an elevated level of tumor suppressor p53. Taken together, this study reveals a novel mechanism for a TRIM28-associated protein stability network and establishes TRIM28 as a potential therapeutic target in cancers where TRIM28 is elevated. Finally, we discuss a potential mechanism of KRAB-ZF gene expression controlled by a regulatory feedback loop of TRIM28-KRAB.
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http://dx.doi.org/10.1093/narcan/zcaa007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380489PMC
June 2020

TMEM9-v-ATPase Activates Wnt/β-Catenin Signaling Via APC Lysosomal Degradation for Liver Regeneration and Tumorigenesis.

Hepatology 2021 02 17;73(2):776-794. Epub 2020 Nov 17.

Department of Experimental Radiation Oncology, Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX.

Background And Aims: How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier.

Approach And Results: TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain β-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of β-catenin, independent of HCC-associated β-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/β-catenin through APC stabilization and β-catenin cytosolic retention.

Conclusions: Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.
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http://dx.doi.org/10.1002/hep.31305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647947PMC
February 2021

Verteporfin Inhibits PD-L1 through Autophagy and the STAT1-IRF1-TRIM28 Signaling Axis, Exerting Antitumor Efficacy.

Cancer Immunol Res 2020 07 7;8(7):952-965. Epub 2020 Apr 7.

Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Programmed cell death 1 ligand 1 (PD-L1) is a key driver of tumor-mediated immune suppression, and targeting it with antibodies can induce therapeutic responses. Given the costs and associated toxicity of PD-L1 blockade, alternative therapeutic strategies are needed. Using reverse-phase protein arrays to assess drugs in use or likely to enter trials, we performed a candidate drug screen for inhibitors of PD-L1 expression and identified verteporfin as a possible small-molecule inhibitor. Verteporfin suppressed basal and IFN-induced PD-L1 expression and through Golgi-related autophagy and disruption of the STAT1-IRF1-TRIM28 signaling cascade, but did not affect the proinflammatory CIITA-MHC II cascade. Within the tumor microenvironment, verteporfin inhibited PD-L1 expression, which associated with enhanced T-lymphocyte infiltration. Inhibition of chromatin-associated enzyme PARP1 induced PD-L1 expression in high endothelial venules (HEV) in tumors and, when combined with verteporfin, enhanced therapeutic efficacy. Thus, verteporfin effectively targets PD-L1 through transcriptional and posttranslational mechanisms, representing an alternative therapeutic strategy for targeting PD-L1.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204534PMC
July 2020

ZEB1/NuRD complex suppresses TBC1D2b to stimulate E-cadherin internalization and promote metastasis in lung cancer.

Nat Commun 2019 11 12;10(1):5125. Epub 2019 Nov 12.

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis.
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http://dx.doi.org/10.1038/s41467-019-12832-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851102PMC
November 2019

Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas.

J Natl Cancer Inst 2020 01;112(1):95-106

Department of Leukemia.

Background: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown.

Methods: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided.

Results: hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients.

Conclusion: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.
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http://dx.doi.org/10.1093/jnci/djz078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489062PMC
January 2020

Cross-talk between chromatin acetylation and SUMOylation of tripartite motif-containing protein 24 (TRIM24) impacts cell adhesion.

J Biol Chem 2018 05 9;293(19):7476-7485. Epub 2018 Mar 9.

Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, Houston, Texas 77030; University of Texas M.D. Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030. Electronic address:

Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers. Numerous interactions between specific reader protein domains and histone PTMs and their regulatory outcomes have been reported, but little is known about how reader proteins may in turn be modulated by these interactions. Tripartite motif-containing protein 24 (TRIM24) is a histone reader aberrantly expressed in multiple cancers. Here, our investigation revealed functional cross-talk between histone acetylation and TRIM24 SUMOylation. Binding of TRIM24 to chromatin via its tandem PHD-bromodomain, which recognizes unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/K23ac), led to TRIM24 SUMOylation at lysine residues 723 and 741. Inactivation of the bromodomain, either by mutation or with a small-molecule inhibitor, IACS-9571, abolished TRIM24 SUMOylation. Conversely, inhibition of histone deacetylation markedly increased TRIM24's interaction with chromatin and its SUMOylation. Of note, gene expression profiling of MCF7 cells expressing WT SUMO-deficient TRIM24 identified cell adhesion as the major pathway regulated by the cross-talk between chromatin acetylation and TRIM24 SUMOylation. In conclusion, our findings establish a new link between histone H3 acetylation and SUMOylation of the reader protein TRIM24, a functional connection that may bear on TRIM24's oncogenic function and may inform future studies of PTM cross-talk between histones and epigenetic regulators.
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http://dx.doi.org/10.1074/jbc.RA118.002233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5950014PMC
May 2018

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes.

BMC Genomics 2018 02 20;19(1):150. Epub 2018 Feb 20.

The Department of Epigenetics and Molecular Carcinogenesis, University of Texas Graduate School of Biomedical Sciences at Houston and The Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, Houston, Texas, 77030, USA.

Background: Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy.

Results: To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells.

Discussion: Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway.

Conclusions: Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.
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http://dx.doi.org/10.1186/s12864-018-4533-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819162PMC
February 2018

Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis.

Genome Res 2018 02 22;28(2):159-170. Epub 2017 Dec 22.

Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences and Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized ("normal") human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif -values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.
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http://dx.doi.org/10.1101/gr.226019.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5793780PMC
February 2018

GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation.

Stem Cell Reports 2018 01 14;10(1):287-299. Epub 2017 Dec 14.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Program in Epigenetics and Molecular Carcinogenesis, The Graduate School of Biomedical Sciences (GSBS) of the University of Texas Health Science Center at Houston, Houston, TX 77030, USA; Program in Genes and Development, The Graduate School of Biomedical Sciences (GSBS) of the University of Texas Health Science Center at Houston, Houston, TX 77030, USA. Electronic address:

Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF) signaling pathway in early embryoid bodies (EBs). Gcn5 EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.
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http://dx.doi.org/10.1016/j.stemcr.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768892PMC
January 2018

p53-independent DUX4 pathology in cell and animal models of facioscapulohumeral muscular dystrophy.

Dis Model Mech 2017 10 28;10(10):1211-1216. Epub 2017 Jul 28.

Lillehei Heart Institute, University of Minnesota, 312 Church St. SE, Minneapolis, MN 55455, USA

Facioscapulohumeral muscular dystrophy (FSHD) is a genetically dominant myopathy caused by mutations that disrupt repression of the normally silent gene, which encodes a transcription factor that has been shown to interfere with myogenesis when misexpressed at very low levels in myoblasts and to cause cell death when overexpressed at high levels. A previous report using adeno-associated virus to deliver high levels of DUX4 to mouse skeletal muscle demonstrated severe pathology that was suppressed on a -knockout background, implying that DUX4 acted through the p53 pathway. Here, we investigate the p53 dependence of DUX4 using various and models. We find that inhibiting p53 has no effect on the cytoxicity of DUX4 on C2C12 myoblasts, and that expression of DUX4 does not lead to activation of the p53 pathway. DUX4 does lead to expression of the classic p53 target gene (p21) but in a p53-independent manner. Meta-analysis of 5 publicly available data sets of DUX4 transcriptional profiles in both human and mouse cells shows no evidence of p53 activation, and further reveals that is a mouse-specific target of DUX4. When the inducible DUX4 mouse model is crossed onto the -null background, we find no suppression of the male-specific lethality or skin phenotypes that are characteristic of the transgene, and find that primary myoblasts from this mouse are still killed by DUX4 expression. These data challenge the notion that the p53 pathway is central to the pathogenicity of DUX4.
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http://dx.doi.org/10.1242/dmm.030064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665455PMC
October 2017

Rapid monoisotopic cisplatin based barcoding for multiplexed mass cytometry.

Sci Rep 2017 06 19;7(1):3779. Epub 2017 Jun 19.

Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously. Existing mass cytometry barcoding approaches require time intensive labeling, reduce the number of biologically meaningful parameters and/or rely on expensive reagents. We present an approach utilizing monoisotopic cisplatin to perform cell barcoding that does not require cell permeabilization, can be completed in 10 minutes and can be utilized in combination with existing barcoding techniques to greatly increase the number of samples which can be multiplexed to improve throughput and consistency.
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http://dx.doi.org/10.1038/s41598-017-03610-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476666PMC
June 2017

Sample Preparation for Mass Cytometry Analysis.

J Vis Exp 2017 04 29(122). Epub 2017 Apr 29.

Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.
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http://dx.doi.org/10.3791/54394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565122PMC
April 2017

Systematic Epigenomic Analysis Reveals Chromatin States Associated with Melanoma Progression.

Cell Rep 2017 04;19(4):875-889

Division of Cancer Medicine, Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. Electronic address:

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.
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http://dx.doi.org/10.1016/j.celrep.2017.03.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473172PMC
April 2017

LncPRESS1 Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56.

Mol Cell 2016 12;64(5):967-981

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Stem Cell and Development Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Cancer Epigenetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA. Electronic address:

Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.
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http://dx.doi.org/10.1016/j.molcel.2016.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137794PMC
December 2016

Bromodomain Histone Readers and Cancer.

J Mol Biol 2017 06 24;429(13):2003-2010. Epub 2016 Nov 24.

Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Stem Cell & Development Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA. Electronic address:

Lysine acetylation of histone proteins is a fundamental post-translational modification that regulates chromatin structure and plays an important role in gene transcription. Aberrant levels of histone lysine acetylation are associated with the development of several diseases. Acetyl-lysine modifications create docking sites for bromodomains, which are structurally conserved modules present in transcription-associated proteins that are termed "reader" proteins. Bromodomain-containing reader proteins are part of multiprotein complexes that regulate transcription programs, which are often associated with profound phenotypic changes. Many bromodomain-containing proteins are aberrantly expressed in diseases, as best studied in cancers, where bromodomain proteins impact the expression of oncogenes and anti-apoptotic proteins. Thus, bromodomain readers of histone acetylation have emerged as attractive targets for cancer drug discovery, prompting immense interest in epigenetic-focused, medicinal chemistry to develop structurally guided chemical probes of bromodomains. Here, we describe bromodomain-containing proteins with defined roles in cancer and highlight recent progress in the development of bromodomain inhibitors.
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http://dx.doi.org/10.1016/j.jmb.2016.11.020DOI Listing
June 2017

Cancer: Acidic shield puts a chink in p53's armour.

Nature 2016 10 14;538(7623):45-46. Epub 2016 Sep 14.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

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http://dx.doi.org/10.1038/nature19469DOI Listing
October 2016

Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer.

Tumour Biol 2016 Nov 16;37(11):14803-14812. Epub 2016 Sep 16.

Tumor Treatment and Prevention Institute, Harbin Medical University, Baojian Road 6, Nangang District, Harbin, Heilongjiang Province, 150081, China.

Acetylated H3 lysine 23 (H3K23ac) is a specific histone post-translational modification recognized by oncoprotein TRIM24. However, it is not clear whether H3K23ac levels are correlated with TRIM24 expression and what role H3K23ac may have in cancer. In this study, we collected breast carcinoma samples from 121 patients and conducted immunohistochemistry to determine the levels of TRIM24 and H3K23ac in breast cancer. Our results demonstrated that TRIM24 expression is positively correlated with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict shorter overall survival of breast cancer patients. We also showed that both TRIM24 and H3K23ac are higher in HER2-positive patients, and their levels were positively correlated with HER2 levels in breast cancer. Moreover, TRIM24 expression is associated with estrogen receptor (ER) and progesterone receptor (PR) statuses in both our cohort and The Cancer Genome Atlas (TCGA) breast carcinoma. In summary, our results revealed an important role of TRIM24 and H3K23ac in breast cancer and provided further evidence that TRIM24 small-molecule inhibitors may benefit ER- and PR-negative or HER2-positive breast cancer patients.
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http://dx.doi.org/10.1007/s13277-016-5344-zDOI Listing
November 2016

Outside the p53 RING: Transcription Regulation by Chromatin-Bound MDM2.

Mol Cell 2016 06;62(6):805-807

Department of Epigenetics & Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Stem Cell & Development Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA. Electronic address:

Evidence mounts, via two studies published in Molecular Cell (Riscal et al., 2016; Wienken et al., 2016), that chromatin-bound MDM2 impacts pluripotency and metabolism to promote survival and proliferation of cancer cells, independently of p53 degradation.
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http://dx.doi.org/10.1016/j.molcel.2016.05.035DOI Listing
June 2016

TRIM-ing Ligand Dependence in Castration-Resistant Prostate Cancer.

Cancer Cell 2016 06;29(6):776-778

Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA. Electronic address:

Prostate cancer is lethal when tumors evolve to activate androgen receptor signaling, which circumvents ligand-deprivation therapy. In this issue of Cancer Cell, Groner et al. show that histone reader and transcription co-regulator TRIM24 occupies a central role in this evolution, nominating inhibitors of TRIM24's bromodomain as a new therapeutic avenue.
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http://dx.doi.org/10.1016/j.ccell.2016.05.014DOI Listing
June 2016

Dual Roles of RNF2 in Melanoma Progression.

Cancer Discov 2015 Dec 8;5(12):1314-27. Epub 2015 Oct 8.

Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas. Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, Texas. Institute for Health Transformation, The University of Texas System, Houston, Texas.

Unlabelled: Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFβ signaling. In contrast, RNF2's oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms.

Significance: The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670809PMC
December 2015

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor.

J Med Chem 2016 Feb 6;59(4):1440-54. Epub 2015 Jul 6.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center , 1515 Holcombe Boulevard , Houston, Texas 77030, United States.

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755933PMC
February 2016

Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Genes Dev 2015 Apr 15;29(8):803-16. Epub 2015 Apr 15.

Center for Cancer Epigenetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA; Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas 78957, USA;

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.
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http://dx.doi.org/10.1101/gad.255109.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403257PMC
April 2015

TRIM24 is a p53-induced E3-ubiquitin ligase that undergoes ATM-mediated phosphorylation and autodegradation during DNA damage.

Mol Cell Biol 2014 Jul;34(14):2695-709

Tumor suppressor p53 protects cells from genomic insults and is a target of mutation in more than 50% of human cancers. Stress-mediated modification and increased stability of p53 promote p53 interaction with chromatin, which results in transcription of target genes that are critical for the maintenance of genomic integrity. We recently discovered that TRIM24, an E3-ubiquitin ligase, ubiquitinates and promotes proteasome-mediated degradation of p53. Here, we show that TRIM24 is destabilized by ATM-mediated phosphorylation of TRIM24S768 in response to DNA damage, which disrupts TRIM24-p53 interactions and promotes the degradation of TRIM24. Transcription of TRIM24 is directly induced by damage-activated p53, which binds p53 response elements and activates expression of TRIM24. Newly synthesized TRIM24 interacts with phosphorylated p53 to target it for degradation and termination of the DNA damage response. These studies indicate that TRIM24, like MDM2, controls p53 levels in an autoregulatory feedback loop. However, unlike MDM2, TRIM24 also targets activated p53 to terminate p53-regulated response to DNA damage.
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http://dx.doi.org/10.1128/MCB.01705-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097665PMC
July 2014

ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression.

Nature 2014 Apr 2;508(7495):263-8. Epub 2014 Mar 2.

1] Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA [2] Center for Cancer Epigenetics, Center for Genetics and Genomics, and Center for Stem Cell and Developmental Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA [3] Genes and Development Graduate Program, The University of Texas Graduate School of Biomedical Sciences, Houston, Teaxs 77030, USA.

Recognition of modified histones by 'reader' proteins plays a critical role in the regulation of chromatin. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies. Here we show that the candidate tumour suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates RNA polymerase II elongation. Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific 'Ser 31' residue in a composite pocket formed by the tandem bromo-PWWP domains of ZMYND11. Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.
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http://dx.doi.org/10.1038/nature13045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4142212PMC
April 2014
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