For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.
Mechanisms and models of somatic cell reprogramming.
Nat Rev Genet. 2013 Jun;14(6):427-39.
Buganim, Y.*, Faddah, D.A.*, and Jaenisch, R.*
Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic-stem-cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodelling the genome, including new insights into the function of MYC, and describe the different phases, markers and emerging models of reprogramming.
A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1.
Science. 2013 May 31;340(6136):1100-6.
Bar-Peled, L.*, Chantranupong, L.*, Cherniack, A.D., Chen, W.W.*, Ottina, K.A.*, Grabiner, B.C., Spear, E.D., Carter, S.L., Meyerson, M., and Sabatini, D.M.*
The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.
Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
Proc Natl Acad Sci U S A. 2013 May 29.
Dang, M.*, Armbruster, N., Miller, M.A., Cermeno, E.*, Hartmann, M., Bell, G.W.*, Root, D.E., Lauffenburger, D.A., Lodish, H.F.*, and Herrlich, A.*
Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-alpha and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-alpha, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-delta is required for neuregulin (NRG) cleavage, and, indeed, PKC-delta phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.
miR-221 redirects precursor B cells to the bone marrow and regulates their residence.
Eur J Immunol. 2013 May 29.
Knoll, M.*, Simmons, S., Bouquet, C., Grun, J.R., and Melchers, F. (2013).
Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR-221 and miR-222. When Pax5 expression commits these progenitors to monopotent pre-B-lymphocytes the two miRNAs are downregulated. Upon transplantation, stem cells and progenitors can home to the bone marrow, while pre-B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline-induced overexpression of either miR-221 or miR-222 in pre-B-I-cells does not revert their monopotency to multipotency. However, upon transplantation miR-221, but not miR-222, transduced pre-B-I cells regain the capacity to home to the bone marrow. Upon subsequent termination of miR-221-expression by removal of doxycycline, the transplanted cells leave the bone marrow again. Microarray analyses identified 25 downregulated miR-221-target genes, which could function to localize phases of B-lymphocyte development in bone marrow before and after commitment.
Keratins control intercellular adhesion involving PKC-alpha-mediated desmoplakin phosphorylation.
J Cell Biol. 2013 May 27;201(5):681-92.
Kroger, C.*, Loschke, F., Schwarz, N., Windoffer, R., Leube, R.E., and Magin, T.M.
Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-alpha-mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-alpha activity is regulated by Rack1-keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-alpha activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.
TALEN-mediated editing of the mouse Y chromosome.
Nat Biotechnol. 2013 May 12.
Wang, H.*, Hu, Y.C.*, Markoulaki, S.*, Welstead, G.G.*, Cheng, A.W.*, Shivalila, C.S.*, Pyntikova, T.*, Dadon, D.B.*, Voytas, D.F., Bogdanove, A.J., Page, D.C.*, and Jaenisch, R.*
The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes-Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
Cell. 2013 May 9;153(4):910-8.
Wang, H.*, Yang, H.*, Shivalila, C.S.*, Dawlaty, M.M.*, Cheng, A.W.*, Zhang, F., and Jaenisch, R.*
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts.
Genes Dev. 2013 Apr 15;27(8):941-54.
Gurtan, A.M., Ravi, A., Rahl, P.B.*, Bosson, A.D., JnBaptiste, C.K., Bhutkar, A., Whittaker, C.A., Young, R.A.*, and Sharp, P.A.
MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.
Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.
Chromosome Res. 2013 Apr;21(2):101-6.
Earnshaw, W.C., Allshire, R.C., Black, B.E., Bloom, K., Brinkley, B.R., Brown, W., Cheeseman, I.M.*, Choo, K.H.A., Copenhaver, G.P., DeLuca, J.G., Desai, A., Diekmann, S., Erhardt, S., Fitzgerald-Hayes, M., Foltz, D., Fukagawa, T., Gassmann, R., Gerlich, D.W., Glover, D.M., Gorbsky, G.J., Harrison, S.C., Heun, P., Hirota, T., Jansen, L.E., Karpen, G., Kops, G.J., Lampson, M.A., Lens, S.M., Losada, A., Luger, K., Maiato, H., Maddox, P.S., Margolis, R.L., Masumoto, H., McAinsh, A.D., Mellone, B.G., Meraldi, P., Musacchio, A., Oegema, K., O'Neill, R.J., Salmon, E.D., Scott, K.C., Straight, A.F., Stukenberg, P.T., Sullivan, B.A., Sullivan, K.F., Sunkel, C.E., Swedlow, J.R., Walczak, C.E., Warburton, P.E., Westermann, S., Willard, H.F., Wordeman, L., Yanagida, M., Yen, T.J., Yoda, K., and Cleveland, D.W.
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
A comparative perspective on lipid storage in animals.
J Cell Sci. 2013 Apr 1;126(Pt 7):1541-52.
Birsoy, K.*, Festuccia, W.T., and Laplante, M.
Lipid storage is an evolutionary conserved process that exists in all organisms from simple prokaryotes to humans. In Metazoa, long-term lipid accumulation is restricted to specialized cell types, while a dedicated tissue for lipid storage (adipose tissue) exists only in vertebrates. Excessive lipid accumulation is associated with serious health complications including insulin resistance, type 2 diabetes, cardiovascular diseases and cancer. Thus, significant advances have been made over the last decades to dissect out the molecular and cellular mechanisms involved in adipose tissue formation and maintenance. Our current understanding of adipose tissue development comes from in vitro cell culture and mouse models, as well as recent approaches to study lipid storage in genetically tractable lower organisms. This Commentary gives a comparative insight into lipid storage in uni- and multi-cellular organisms with a particular emphasis on vertebrate adipose tissue. We also highlight the molecular mechanisms and nutritional signals that regulate the formation of mammalian adipose tissue.
*Author affiliated with Whitehead Institute for Biomedical Research