Scientific Papers

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

Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.

Nat Protoc. 2014 Aug;9(8):1956-68.

Yang, H.*, Wang, H.*, and Jaenisch, R.*

Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.

 

iPipet: sample handling using a tablet.

Nat Methods. 2014 Jul 30;11(8):784-785.

Zielinski, D., Gordon, A., Zaks, B.L., and Erlich, Y.

 

Depletion of RICTOR, an essential protein component of mTORC2, decreases male lifespan.

Aging Cell. 2014 Jul 25.

Lamming, D.W.*, Mihaylova, M.M.*, Katajisto, P.*, Baar, E.L., Yilmaz, O.H.*, Hutchins, A.*, Gultekin, Y.*, Gaither, R.*, and Sabatini, D.M.*

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR), robustly extends the lifespan of model organisms including mice. We recently found that chronic treatment with rapamycin not only inhibits mTOR complex 1 (mTORC1), the canonical target of rapamycin, but also inhibits mTOR complex 2 (mTORC2) in vivo. While genetic evidence strongly suggests that inhibition of mTORC1 is sufficient to promote longevity, the impact of mTORC2 inhibition on mammalian longevity has not been assessed. RICTOR is a protein component of mTORC2 that is essential for its activity. We examined three different mouse models of Rictor loss: mice heterozygous for Rictor, mice lacking hepatic Rictor, and mice in which Rictor was inducibly deleted throughout the body in adult animals. Surprisingly, we find that depletion of RICTOR significantly decreases male, but not female, lifespan. While the mechanism by which RICTOR loss impairs male survival remains obscure, we find that the effect of RICTOR depletion on lifespan is independent of the role of hepatic mTORC2 in promoting glucose tolerance. Our results suggest that inhibition of mTORC2 signaling is detrimental to males, which may explain in part why interventions that decrease mTOR signaling show greater efficacy in females.

 

The extreme anterior domain is an essential craniofacial organizer acting through Kinin-Kallikrein signaling.

Cell Rep. 2014 Jul 24;8(2):596-609.

Jacox, L.*, Sindelka, R.*, Chen, J.*, Rothman, A.*, Dickinson, A.*, and Sive, H.*

The extreme anterior domain (EAD) is a conserved embryonic region that includes the presumptive mouth. We show that the Kinin-Kallikrein pathway is active in the EAD and necessary for craniofacial development in Xenopus and zebrafish. The mouth failed to form and neural crest (NC) development and migration was abnormal after loss of function (LOF) in the pathway genes kng, encoding Bradykinin (xBdk), carboxypeptidase-N (cpn), which cleaves Bradykinin, and neuronal nitric oxide synthase (nNOS). Consistent with a role for nitric oxide (NO) in face formation, endogenous NO levels declined after LOF in pathway genes, but these were restored and a normal face formed after medial implantation of xBdk-beads into LOF embryos. Facial transplants demonstrated that Cpn function from within the EAD is necessary for the migration of first arch cranial NC into the face and for promoting mouth opening. The study identifies the EAD as an essential craniofacial organizer acting through Kinin-Kallikrein signaling.

 

Polo-like kinase 1 licenses CENP-A deposition at centromeres.

Cell. 2014 Jul 17;158(2):397-411.

McKinley, K.L.*, and Cheeseman, I.M.*

To ensure the stable transmission of the genome during vertebrate cell division, the mitotic spindle must attach to a single locus on each chromosome, termed the centromere. The fundamental requirement for faithful centromere inheritance is the controlled deposition of the centromere-specifying histone, CENP-A. However, the regulatory mechanisms that ensure the precise control of CENP-A deposition have proven elusive. Here, we identify polo-like kinase 1 (Plk1) as a centromere-localized regulator required to initiate CENP-A deposition in human cells. We demonstrate that faithful CENP-A deposition requires integrated signals from Plk1 and cyclin-dependent kinase (CDK), with Plk1 promoting the localization of the key CENP-A deposition factor, the Mis18 complex, and CDK inhibiting Mis18 complex assembly. By bypassing these regulated steps, we uncoupled CENP-A deposition from cell-cycle progression, resulting in mitotic defects. Thus, CENP-A deposition is controlled by a two-step regulatory paradigm comprised of Plk1 and CDK that is crucial for genomic integrity.

 

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

Cell. 2014 Jul 17;158(2):434-48.

Taipale, M.*, Tucker, G., Peng, J., Krykbaeva, I.*, Lin, Z.Y., Larsen, B., Choi, H., Berger, B., Gingras, A.C., and Lindquist, S.*

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct beta-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement.

 

GPR107, a GPCR essential for intoxication by P. aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

J Biol Chem. 2014 Jul 16.

Tafesse, F.G.*, Guimaraes, C.P.*, Maruyama, T.*, Carette, J.E., Lory, S., Brummelkamp, T.R., and Ploegh, H.L.*

A number of toxins, including Exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan GPCR. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.

 

Sox2: Masterminding the root of cancer.

Cancer Cell. 2014 Jul 14;26(1):3-5.

Tam, W.L.*, and Ng, H.H.*

The transcription factor Sox2 is a master regulator that maintains stemness in embryonic stem cells and neural stem cells. Using elegant lineage tracing strategies and genetic reporter mouse models, two studies (one of which is by Vanner and colleagues in this issue of Cancer Cell) now demonstrate that rare Sox2-expressing cells are the founding cancer stem cell population driving tumor initiation and therapy resistance.

 

Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

Nature. 2014 Jun 22.

Kwiatkowski, N.*, Zhang, T.*, Rahl, P.B.*, Abraham, B.J.*, Reddy, J.*, Ficarro, S.B., Dastur, A., Amzallag, A., Ramaswamy, S., Tesar, B., Jenkins, C.E., Hannett, N.M.*, McMillin, D., Sanda, T., Sim, T., Kim, N.D., Look, T., Mitsiades, C.S., Weng, A.P., Brown, J.R., Benes, C.H., Marto, J.A., Young, R.A.*, and Gray, N.S.

Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.

 

Detecting coevolution in mammalian sperm-egg fusion proteins.

Mol Reprod Dev. 2014 Jun;81(6):531-8.

Claw, K.G., George, R.D.*, and Swanson, W.J.

Interactions between sperm and egg proteins can occur physically between gamete surface-binding proteins, and genetically between gamete proteins that work in complementary pathways in which they may not physically interact. Physically interacting sperm-egg proteins have been functionally identified in only a few species, and none have been verified within mammals. Candidate genes on both the sperm and egg surfaces exist, but gene deletion studies do not support functional interactions between these sperm-egg proteins; interacting sperm-egg proteins thus remain elusive. Cooperative gamete proteins undergo rapid evolution, and it is predicted that these sperm-egg proteins will also have correlated evolutionary rates due to compensatory changes on both the sperm and egg. To explore potential physical and genetic interactions in sperm-egg proteins, we sequenced four candidate genes from diverse primate species, and used regression and likelihood methods to test for signatures of coevolution between sperm-egg gene pairs. With both methods, we found that the egg protein CD9 coevolves with the sperm protein IZUMO1, suggesting a physical or genetic interaction occurs between them. With regression analysis, we found that CD9 and CRISP2 have correlated rates of evolution, and with likelihood analysis, that CD9 and CRISP1 have correlated rates. This suggests that the different tests may reflect different levels of interaction, be it physical or genetic. Coevolution tests thus provide an exploratory method for detecting potentially interacting sperm-egg protein pairs. (C) 2014 Wiley Periodicals, Inc.

 

 
*Author affiliated with Whitehead Institute for Biomedical Research

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