Scientific Papers

For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.

Calcineurin determines toxic versus beneficial responses to alpha-synuclein.

Proc Natl Acad Sci U S A. 2014 Aug 26;111(34):E3544-52.

Caraveo, G.*, Auluck, P.K.*, Whitesell, L.*, Chung, C.Y.*, Baru, V., Mosharov, E.V., Yan, X., Ben-Johny, M., Soste, M., Picotti, P., Kim, H., Caldwell, K.A., Caldwell, G.A., Sulzer, D., Yue, D.T., and Lindquist, S.*

Calcineurin (CN) is a highly conserved Ca2+-calmodulin (CaM)-dependent phosphatase that senses Ca2+ concentrations and transduces that information into cellular responses. Ca2+ homeostasis is disrupted by alpha-synuclein (alpha-syn), a small lipid binding protein whose misfolding and accumulation is a pathological hallmark of several neurodegenerative diseases. We report that alpha-syn, from yeast to neurons, leads to sustained highly elevated levels of cytoplasmic Ca2+, thereby activating a CaM-CN cascade that engages substrates that result in toxicity. Surprisingly, complete inhibition of CN also results in toxicity. Limiting the availability of CaM shifts CN's spectrum of substrates toward protective pathways. Modulating CN or CN's substrates with highly selective genetic and pharmacological tools (FK506) does the same. FK506 crosses the blood brain barrier, is well tolerated in humans, and is active in neurons and glia. Thus, a tunable response to CN, which has been conserved for a billion years, can be targeted to rebalance the phosphatase's activities from toxic toward beneficial substrates. These findings have immediate therapeutic implications for synucleinopathies.

 

PyBamView: a browser based application for viewing short read alignments.

Gymrek, M.*

Bioinformatics. 2014 Aug 21.

Current sequence alignment browsers allow visualization of large and complex next-generation sequencing datasets. However, most of these tools provide inadequate display of insertions and can be cumbersome to use on large datasets. I implemented PyBamView, a lightweight web application for visualizing short read alignments. It provides an easy-to-use web interface for viewing alignments across multiple samples, with a focus on accurate visualization of insertions. Availability and Implementation: PyBamView is available as a standard python package. The source code is freely available under the MIT license at https://mgymrek.github.io/pybamview.

 

The landscape of human STR variation.

Genome Res. 2014 Aug 18.

Willems, T.F.*, Gymrek, M.*, Highnam, G., Mittelman, D., and Erlich, Y.*

Genome ResShort tandem repeats are among the most polymorphic loci in the human genome. These loci play a role in the etiology of a range of genetic diseases and have been frequently utilized in forensics, population genetics, and genetic genealogy. Despite this plethora of applications, little is known about the variation of most STRs in the human population. Here, we report the largest-scale analysis of human STR variation to date. We collected information for nearly 700,000 STR loci across over 1,000 individuals in phase 1 of the 1000 Genomes Project. Extensive quality controls show that reliable allelic spectra can be obtained for close to 90% of the STR loci in the genome. We utilize this call set to analyze determinants of STR variation, assess the human reference genome's representation of STR alleles, find STR loci with common loss-of-function alleles, and obtain initial estimates of the linkage disequilibrium between STRs and common SNPs. Overall, these analyses further elucidate the scale of genetic variation beyond classical point mutations.

 

Contrasting roles of histone 3 lysine 27 demethylases in acute lymphoblastic leukaemia.

Nature. 2014 Aug 17.

Ntziachristos, P., Tsirigos, A., Welstead, G.G.*, Trimarchi, T., Bakogianni, S., Xu, L., Loizou, E., Holmfeldt, L., Strikoudis, A., King, B., Mullanders, J., Becksfort, J., Nedjic, J., Paietta, E., Tallman, M.S., Rowe, J.M., Tonon, G., Satoh, T., Kruidenier, L., Prinjha, R., Akira, S., Van Vlierberghe, P., Ferrando, A.A., Jaenisch, R.*, Mullighan, C.G., and Aifantis, I.

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.

 

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.

Genes Dev. 2014 Aug 15.

Sexton, A.N., Regalado, S.G., Lai, C.S.*, Cost, G.J., O'Neil, C.M., Urnov, F.D., Gregory, P.D., Jaenisch, R.*, Collins, K., and Hockemeyer, D.

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.

 

From yeast to patient neurons and back again: A powerful new discovery platform.

Mov Disord. 2014 Aug 14.

Tardiff, D.F.*, Khurana, V.*, Chung, C.Y.*, and Lindquist, S.*

No disease-modifying therapies are available for synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). The lack of therapies has been impeded by a paucity of validated drug targets and problematic cell-based model systems. New approaches are therefore needed to identify genes and compounds that directly target the underlying cellular pathologies elicited by the pathological protein, alpha-synuclein (alpha-syn). This small, lipid-binding protein impinges on evolutionarily conserved processes such as vesicle trafficking and mitochondrial function. For decades, the genetically tractable, single-cell eukaryote, budding yeast, has been used to study nearly all aspects of cell biology. More recently, yeast has revealed key insights into the underlying cellular pathologies caused by alpha-syn. The robust cellular toxicity caused by alpha-syn expression facilitates unbiased high-throughput small-molecule screening. Critically, one must validate the discoveries made in yeast in disease-relevant neuronal models. Here, we describe two recent reports that together establish a yeast-to-human discovery platform for synucleinopathies. In this exemplar, genes and small molecules identified in yeast were validated in patient-derived neurons that present the same cellular phenotypes initially discovered in yeast. On validation, we returned to yeast, where unparalleled genetic approaches facilitated the elucidation of a small molecule's mode of action. This approach enabled the identification and neuronal validation of a previously unknown "druggable" node that interfaces with the underlying, precipitating pathologies caused by alpha-syn. Such platforms can provide sorely needed leads and fresh ideas for disease-modifying therapy for these devastating diseases. (c) 2014 International Parkinson and Movement Disorder Society.

 

Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells.

Oncogene. 2014 Aug 11.

Watnick, R.S., Rodriguez, R.K., Wang, S., Blois, A.L., Rangarajan, A.*, Ince, T.*, and Weinberg, R.A.*

Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation.

 

The cell's view of animal body-plan evolution.

Integr Comp Biol. 2014 Aug 8

Lyons, D.C., Martindale, M.Q., and Srivastava, M.*

An adult animal's form is shaped by the collective behavior of cells during embryonic development. To understand the forces that drove the divergence of animal body-plans, evolutionary developmental biology has focused largely on studying genetic networks operating during development. However, it is less well understood how these networks modulate characteristics at the cellular level, such as the shape, polarity, or migration of cells. We organized the "Cell's view of animal body plan evolution" symposium for the 2014 The Society for Integrative and Comparative Biology meeting with the explicit goal of bringing together researchers studying the cell biology of embryonic development in diverse animal taxa. Using a broad range of established and emerging technologies, including live imaging, single-cell analysis, and mathematical modeling, symposium participants revealed mechanisms underlying cells' behavior, a few of which we highlight here. Shape, adhesion, and movements of cells can be modulated over the course of evolution to alter adult body-plans and a major theme explored during the symposium was the role of actomyosin in coordinating diverse behaviors of cells underlying morphogenesis in a myriad of contexts. Uncovering whether conserved or divergent genetic mechanisms guide the contractility of actomyosin in these systems will be crucial to understanding the evolution of the body-plans of animals from a cellular perspective. Many speakers presented research describing developmental phenomena in which cell division and tissue growth can control the form of the adult, and other presenters shared work on studying cell-fate specification, an important source of novelty in animal body-plans. Participants also presented studies of regeneration in annelids, flatworms, acoels, and cnidarians, and provided a unifying view of the regulation of cellular behavior during different life-history stages. Additionally, several presentations highlighted technological advances that glean mechanistic insights from new and emerging model systems, thereby providing the phylogenetic breadth so essential for studying animal evolution. Thus, we propose that an explicit study of cellular phenomena is now possible for a wide range of taxa, and that it will be highly informative for understanding the evolution of animal body-plans.

 

Retinoic adcid activates two pathways required for meiosis in mice.

PLoS Genet. 2014 Aug 7;10(8):e1004541.

Koubova, J.*, Hu, Y.C., Bhattacharyya, T., Soh, Y.Q., Gill, M.E., Goodheart, M.L., Hogarth, C.A., Griswold, M.D., and Page, D.C.*

In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA), the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function.

 

Secretion of circular proteins using sortase.

Methods Mol Biol. 2014;1174:73-83.

Strijbis, K., and Ploegh, H.L.*

Circular proteins occur naturally and have been found in microorganisms, plants, and eukaryotes where they are commonly involved in host defense. Properties of circular proteins include enhanced resistance to exoproteases, increased thermostability, longer life spans, and increased activity. Using an enzymatic approach based on the bacterial sortase A (SrtA) transpeptidase, N- and C-termini of conventional linear proteins can be linked resulting in a circular protein. Circularization of bioengineered linear substrate proteins can indeed confer the desirable properties associated with circular proteins. Here, we describe how cells can be manipulated to secrete circularized proteins for substrates of choice via sortase-mediated circularization in the lumen of the endoplasmic reticulum.

 
*Author affiliated with Whitehead Institute for Biomedical Research

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