Scientific Papers

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

Nutrient-sensing mechanisms across evolution.

Cell. 2015 Mar 26;161(1):67-83.

Chantranupong, L.*, Wolfson, R.L.*, and Sabatini, D.M.*

For organisms to coordinate their growth and development with nutrient availability, they must be able to sense nutrient levels in their environment. Here, we review select nutrient-sensing mechanisms in a few diverse organisms. We discuss how these mechanisms reflect the nutrient requirements of specific species and how they have adapted to the emergence of multicellularity in eukaryotes.

 

Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions.

Proc Natl Acad Sci U S A. 2015 Mar 24;112(12):3841-6.

Ji, X.*, Dadon, D.B.*, Abraham, B.J.*, Lee, T.I.*, Jaenisch, R.*, Bradner, J.E., and Young, R.A.*

More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call "chromatin proteomic profiling," to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types.

 

Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.

Nat Biotechnol. 2015 Mar 23.

Maruyama, T.*, Dougan, S.K.*, Truttmann, M.C.*, Bilate, A.M.*, Ingram, J.R.*, and Ploegh, H.L.*

Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator-like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.

 

Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers.

Mol Cell. 2015 Mar 18. pii: S1097-2765(15)00128-8.

Hnisz, D.*, Schuijers, J.*, Lin, C.Y., Weintraub, A.S.*, Abraham, B.J.*, Lee, T.I.*, Bradner, J.E., and Young, R.A.*

Super-enhancers and stretch enhancers (SEs) drive expression of genes that play prominent roles in normal and disease cells, but the functional importance of these clustered enhancer elements is poorly understood, so it is not clear why genes key to cell identity have evolved regulation by such elements. Here, we show that SEs consist of functional constituent units that concentrate multiple developmental signaling pathways at key pluripotency genes in embryonic stem cells and confer enhanced responsiveness to signaling of their associated genes. Cancer cells frequently acquire SEs at genes that promote tumorigenesis, and we show that these genes are especially sensitive to perturbation of oncogenic signaling pathways. Super-enhancers thus provide a platform for signaling pathways to regulate genes that control cell identity during development and tumorigenesis.

 

Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.

Oncogene. 2015 Mar 16.

Strohecker, A.M., Joshi, S., Possemato, R.*, Abraham, R.T., Sabatini, D.M.*, and White, E.

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based short hairpin RNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.Oncogene advance online publication, 16 March 2015; doi:10.1038/onc.2015.23.

 

Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor.

Science. 2015 Mar 6;347(6226):1113-7.

Burg, J.S., Ingram, J.R.*, Venkatakrishnan, A.J., Jude, K.M., Dukkipati, A., Feinberg, E.N., Angelini, A., Waghray, D., Dror, R.O., Ploegh, H.L.*, and Garcia, K.C.

Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor's inactive state.

 

Licensing of primordial germ cells for gametogenesis depends on genital ridge signaling.

PLoS Genet. 2015 Mar 4;11(3):e1005019.

Hu, Y.C.*, Nicholls, P.K.*, Soh, Y.Q.*, Daniele, J.R., Junker, J.P., van Oudenaarden, A., and Page, D.C.*

In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous and gonad-independent event, based on observations that some PGCs, having migrated not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel to ovarian germ cells -- and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4 conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.

 

A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth.

Genes Dev. 2015 Mar 1;29(5):483-8.

Meacham, C.E., Lawton, L.N.*, Soto-Feliciano, Y.M., Pritchard, J.R., Joughin, B.A., Ehrenberger, T., Fenouille, N., Zuber, J., Williams, R.T., Young, R.A.*, and Hemann, M.T.

We performed a genome-scale shRNA screen for modulators of B-cell leukemia progression in vivo. Results from this work revealed dramatic distinctions between the relative effects of shRNAs on the growth of tumor cells in culture versus in their native microenvironment. Specifically, we identified many "context-specific" regulators of leukemia development. These included the gene encoding the zinc finger protein Phf6. While inactivating mutations in PHF6 are commonly observed in human myeloid and T-cell malignancies, we found that Phf6 suppression in B-cell malignancies impairs tumor progression. Thus, Phf6 is a "lineage-specific" cancer gene that plays opposing roles in developmentally distinct hematopoietic malignancies.

 

Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica.

Metab Eng. 2015 Feb 27;29:56-65.

Qiao, K., Imam Abidi, S.H., Liu, H., Zhang, H., Chakraborty, S., Watson, N.*, Kumaran Ajikumar, P., and Stephanopoulos, G.

Conversion of carbohydrates to lipids at high yield and productivity is essential for cost-effective production of renewable biodiesel. Although some microorganisms can convert sugars to oils, conversion yields and rates are typically low due primarily to allosteric inhibition of the lipid biosynthetic pathway by saturated fatty acids. By reverse engineering the mammalian cellular obese phenotypes, we identified the delta-9 stearoyl-CoA desaturase (SCD) as a rate limiting step and target for the metabolic engineering of the lipid synthesis pathway in Yarrowia lipolytica. Simultaneous overexpression of SCD, Acetyl-CoA carboxylase (ACC1), and Diacylglyceride acyl-transferase (DGA1) in Y. lipolytica yielded an engineered strain exhibiting highly desirable phenotypes of fast cell growth and lipid overproduction including high carbon to lipid conversion yield (84.7% of theoretical maximal yield), high lipid titers (approximately 55g/L), and enhanced tolerance to glucose and cellulose-derived sugars. Moreover, the engineered strain shows a three-fold growth advantage over the wild type strain. As a result, a maximal lipid productivity of approximately 1g/L/h is obtained during the stationary phase. Furthermore, we show that the engineered yeast required cytoskeleton remodeling in eliciting the obesity phenotype. Altogether, our work describes the development of a microbial catalyst with the highest reported lipid yield, titer and productivity to date. This is an important step towards the development of an efficient and cost-effective process for biodiesel production from renewable resources.

 

One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism.

ACS Chem Biol. 2015 Feb 20;10(2):460-5.

Swee, L.K.*, Lourido, S.*, Bell, G.W.*, Ingram, J.R.*, and Ploegh, H.L.*

Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells.

 

 

*Author affiliated with Whitehead Institute for Biomedical Research

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