For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.
Fundamental differences in endoreplication in mammals and Drosophila revealed by analysis of endocycling and endomitotic cells.
Proc Natl Acad Sci U S A. 2013 Apr 23.
Sher, N.*, Von Stetina, J.R.*, Bell, G.W.*, Matsuura, S., Ravid, K., and Orr-Weaver, T.L.*
Throughout the plant and animal kingdoms specific cell types become polyploid, increasing their DNA content to attain a large cell size. In mammals, megakaryocytes (MKs) become polyploid before fragmenting into platelets. The mammalian trophoblast giant cells (TGCs) exploit their size to form a barrier between the maternal and embryonic tissues. The mechanism of polyploidization has been investigated extensively in Drosophila, in which a modified cell cycle-the endocycle, consisting solely of alternating S and gap phases-produces polyploid tissues. During S phase in the Drosophila endocycle, heterochromatin and specific euchromatic regions are underreplicated and reduced in copy number. Here we investigate the properties of polyploidization in murine MKs and TGCs. We induced differentiation of primary MKs and directly microdissected TGCs from embryonic day 9.5 implantation sites. The copy number across the genome was analyzed by array-based comparative genome hybridization. In striking contrast to Drosophila, the genome was uniformly and integrally duplicated in both MKs and TGCs. This was true even for heterochromatic regions analyzed by quantitative PCR. Underreplication of specific regions in polyploid cells is proposed to be due to a slower S phase, resulting from low expression of S-phase genes, causing failure to duplicate late replicating genomic intervals. We defined the transcriptome of TGCs and found robust expression of S-phase genes. Similarly, S-phase gene expression is not repressed in MKs, providing an explanation for the distinct endoreplication parameters compared with Drosophila. Consistent with TGCs endocycling rather than undergoing endomitosis, they have low expression of M-phase genes.
MiR-150 blocks MLL-AF9 associated leukemia by repressing multiple oncogenes.
Mol Cancer Res. 2013 Apr 19.
Bousquet, M.*, Zhuang, G., Meng, C., Ying, W., Cheruku, P.S., Shie, A.T.*, Wang, S., Ge, G., Wong, P.*, Wang, G., Safe, S., and Zhou, B.
The microRNA miR-150, a critical regulator of hematopoiesis, is downregulated in MLL-associated leukemias. In this study, we demonstrate that miR-150 acts as a potent leukemic tumor suppressor by blocking the oncogenic properties of leukemic cells. By using murine MLL-AF9 transformed cells, we demonstrate that miR-150 ectopic expression inhibits blast colony formation and cell growth and increases apoptosis in vitro. More importantly, ectopic expression of miR-150 in transformed MLL-AF9 cells completely blocked the development of myeloid leukemia in transplanted mice. Gene expression profiles demonstrated that miR-150 altered the expression levels of more than 30 "stem cell signature" genes and many others that are involved in cancer pathways. Furthermore, we identified Cbl and Egr2, additional to the known target Myb, as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the pro-apoptotic effects observed in leukemic cells with miR-150 ectopic expression. In conclusion, we demonstrate that miR-150 is a potent leukemic tumor suppressor that regulates multiple oncogenes, providing new key players for the development of therapeutic strategies to treat MLL-AF9 related leukemia.
Intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins.
Proc Natl Acad Sci U S A. 2013 Apr 16;110(16):6406-11.
Tafesse, F.G.*, Sanyal, S.*, Ashour, J.*, Guimaraes, C.P.*, Hermansson, M., Somerharju, P., and Ploegh, H.L.*
Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or blocked in their synthesis pharmacologically through exposure to a serine palmitoyltransferase inhibitor (myriocin) show strongly reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the cell surface was assessed by accessibility of HA on intact cells to exogenously added trypsin and to HA-specific antibodies. Rates of de novo synthesis of viral proteins in wild-type and SGMS1-deficient cells were equivalent, and HA negotiated the intracellular trafficking pathway through the Golgi normally. We engineered a strain of influenza virus to allow site-specific labeling of HA and NA using sortase. Accessibility of both HA and NA to sortase was blocked in SGMS1-deficient cells and in cells exposed to myriocin, with a corresponding inhibition of the release of virus particles from infected cells. Generation of influenza virus particles thus critically relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of a composition compatible with virus budding and release.
Characterization of Torin2, an ATP-competitive inhibitor of mTOR, ATM, and ATR.
Cancer Res. 2013 Apr 15;73(8):2574-2586.
Liu, Q., Xu, C., Kirubakaran, S., Zhang, X., Hur, W., Liu, Y., Kwiatkowski, N.P., Wang, J., Westover, K.D., Gao, P., Ercan, D., Niepel, M., Thoreen, C.C., Kang, S.A.*, Patricelli, M.P., Wang, Y., Tupper, T., Altabef, A., Kawamura, H., Held, K.D., Chou, D.M., Elledge, S.J., Janne, P.A., Wong, K.K., Sabatini, D.M.*, and Gray, N.S.
mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC50 of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC50, 28 nmol/L), ATR (EC50, 35 nmol/L), and DNA-PK (EC50, 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.
Master transcription factors and mediator establish super-enhancers at key cell identity genes.
Cell. 2013 Apr 11;153(2):307-19.
Whyte, W.A.*, Orlando, D.A.*, Hnisz, D.*, Abraham, B.J.*, Lin, C.Y.*, Kagey, M.H.*, Rahl, P.B.*, Lee, T.I.*, and Young, R.A.*
Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity.
Selective inhibition of tumor oncogenes by disruption of super-enhancers.
Cell. 2013 Apr 11;153(2):320-34.
Loven, J.*, Hoke, H.A.*, Lin, C.Y.,* Lau, A.*, Orlando, D.A.*, Vakoc, C.R., Bradner, J.E., Lee, T.I.*, and Young, R.A.*
Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types.
CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state.
J Cell Biol. 2013 Apr 1;201(1):23-32.
Gascoigne, K.E.*, and Cheeseman, I.M.*
Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex. Prior work has identified more than 100 different kinetochore components in human cells. However, little is known about the regulatory processes that specify their assembly upon mitotic entry and disassembly at mitotic exit. In this paper, we used a live-cell imaging-based assay to quantify kinetochore disassembly kinetics and systematically analyze the role of potential regulatory mechanisms in controlling kinetochore assembly state. We find that kinetochore assembly and disassembly was driven primarily by mitotic phosphorylation downstream of cyclin-dependent kinase (CDK). In addition, we demonstrate that nuclear exclusion of the Ndc80 complex helped restrict kinetochore formation to mitosis. Combining constitutive CDK-dependent phosphorylation of CENP-T and forced nuclear localization of the Ndc80 complex partially prevented kinetochore disassembly at mitotic exit and led to chromosome segregation defects in subsequent divisions. In total, we find that the coordinated temporal regulation of outer kinetochore assembly is essential for accurate cell division.
Heritable remodeling of yeast multicellularity by an environmentally responsive prion.
Cell. 2013 Mar 28;153(1):153-65.
Holmes, D.L., Lancaster, A.K.*, Lindquist, S.*, and Halfmann, R.
Prion proteins undergo self-sustaining conformational conversions that heritably alter their activities. Many of these proteins operate at pivotal positions in determining how genotype is translated into phenotype. But the breadth of prion influences on biology and their evolutionary significance are just beginning to be explored. We report that a prion formed by the Mot3 transcription factor, [MOT3(+)], governs the acquisition of facultative multicellularity in the budding yeast Saccharomyces cerevisiae. The traits governed by [MOT3(+)] involved both gains and losses of Mot3 regulatory activity. [MOT3(+)]-dependent expression of FLO11, a major determinant of cell-cell adhesion, produced diverse lineage-specific multicellular phenotypes in response to nutrient deprivation. The prions themselves were induced by ethanol and eliminated by hypoxia-conditions that occur sequentially in the natural respiro-fermentative cycles of yeast populations. These data demonstrate that prions can act as environmentally responsive molecular determinants of multicellularity and contribute to the natural morphological diversity of budding yeast.
Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus.
J Biol Chem. 2013 Mar 22;288(12):8061-73.
Hendricks, G.L., Weirich, K.L., Viswanathan, K., Li, J., Shriver, Z.H., Ashour, J.*, Ploegh, H.L.*, Kurt-Jones, E.A., Fygenson, D.K., Finberg, R.W., Comolli, J.C., and Wang, J.P.
Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.
A novel single-cell screening platform reveals proteome plasticity during yeast stress responses.
J Cell Biol. 2013 Mar 18;200(6):839-50.
Breker, M., Gymrek, M.*, and Schuldiner, M.
Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique "fingerprint" of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome).
*Author affiliated with Whitehead Institute for Biomedical Research