Scientific Papers

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

RNA circularization diminishes immunogenicity and can extend translation duration in vivo.

Mol Cell. 2019 May 2;74(3):508-520.e4. doi: 10.1016/j.molcel.2019.02.015. 

Wesselhoeft, R.A., Kowalski, P.S., Parker-Hale, F.C., Huang, Y., Bisaria, N.*, and Anderson, D.G.

Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

 

Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells.

Proc Natl Acad Sci U S A. 2019 Apr 9;116(15):7353-7362. doi: 10.1073/pnas.1812876116.

Kroger, C.*, Afeyan, A., Mraz, J., Eaton, E.N.*, Reinhardt, F.*, Khodor, Y.L., Thiru, P.*, Bierie, B., Ye, X.*, Burge, C.B., Weinberg, R.A.*

Carcinoma cells residing in an intermediate phenotypic state along the epithelial-mesenchymal (E-M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104(+)/CD44(hi) antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably-both in vitro and in vivo-in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E-M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E-M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.

 

Rate of progression through a continuum of transit-amplifying progenitor cell states regulates blood cell production

Dev Cell. 2019 Apr 8;49(1):118-129.e7. doi: 10.1016/j.devcel.2019.01.026. 

Li, H.*, Natarajan, A.*, Ezike, J., Barrasa, M.I.*, Le, Y.*, Feder, Z.A.*, Yang, H.*, Ma, C., Markoulaki, S.*, and Lodish, H.F.*

The nature of cell-state transitions during the transit-amplifying phases of many developmental processes-hematopoiesis in particular-is unclear. Here, we use single-cell RNA sequencing to demonstrate a continuum of transcriptomic states in committed transit-amplifying erythropoietic progenitors, which correlates with a continuum of proliferative potentials in these cells. We show that glucocorticoids enhance erythrocyte production by slowing the rate of progression through this developmental continuum of transit-amplifying progenitors, permitting more cell divisions prior to terminal erythroid differentiation. Mechanistically, glucocorticoids prolong expression of genes that antagonize and slow induction of genes that drive terminal erythroid differentiation. Erythroid progenitor daughter cell pairs have similar transcriptomes with or without glucocorticoid stimulation, indicating largely symmetric cell division. Thus, the rate of progression along a developmental continuum dictates the absolute number of erythroid cells generated from each transit-amplifying progenitor, suggesting a paradigm for regulating the total output of differentiated cells in numerous other developmental processes.

 

Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements.

Nat Genet. 2019 Apr;51(4):705-715. doi: 10.1038/s41588-019-0360-8.

Ly, P., Brunner, S.F., Shoshani, O., Kim, D.H., Lan, W., Pyntikova, T.*, Flanagan, A.M., Behjati, S., Page, D.C.*, Campbell, P.J., and Cleveland, D.W.

Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed CEN-SELECT, to systematically interrogate the structural landscape of mis-segregated chromosomes. We show that single-chromosome mis-segregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that mis-segregated chromosomes exhibit 120-fold-higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.

 

PRX9 and PRX40 are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity during Arabidopsis anther development.

Plant Cell. 2019 Apr;31(4):848-861. doi: 10.1105/tpc.18.00907. 

Jacobowitz, J.R.*, Doyle, W.C.*, and Weng, J.K.*

Pollen and microspore development are essential steps in the life cycle of all land plants that generate male gametes. Within flowering plants, pollen development occurs inside of the anther. Here, we report the identification of two class III peroxidase-encoding genes, PEROXIDASE9 and PEROXIDASE40, that are genetically redundant and essential for proper anther and pollen development in Arabidopsis thaliana. Arabidopsis double mutants devoid of functional PRX9 and PRX40 are male-sterile. The mutant anthers display swollen, hypertrophic tapetal cells and pollen grains, suggesting disrupted cell wall integrity. These phenotypes lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 likely cross-link extensins to contribute to tapetal cell wall integrity during anther development. This work suggests that PRX9 and PRX40 encode Arabidopsis extensin peroxidases and highlights the importance of extensin cross-linking during pollen development.

 

Acoel genome reveals the regulatory landscape of whole-body regeneration.

Science. 2019 Mar 15;363(6432). pii: eaau6173. doi: 10.1126/science.aau6173.

Gehrke, A.R., Neverett, E., Luo, Y.J., Brandt, A., Ricci, L., Hulett, R.E., Gompers, A., Ruby, J.G., Rokhsar, D.S., Reddien, P.W.*, and Srivastava, M.

Whole-body regeneration is accompanied by complex transcriptomic changes, yet the chromatin regulatory landscapes that mediate this dynamic response remain unexplored. To decipher the regulatory logic that orchestrates regeneration, we sequenced the genome of the acoel worm Hofstenia miamia, a highly regenerative member of the sister lineage of other bilaterians. Epigenomic profiling revealed thousands of regeneration-responsive chromatin regions and identified dynamically bound transcription factor motifs, with the early growth response (EGR) binding site as the most variably accessible during Hofstenia regeneration. Combining egr inhibition with chromatin profiling suggests that Egr functions as a pioneer factor to directly regulate early wound-induced genes. The genetic connections inferred by this approach allowed the construction of a gene regulatory network for whole-body regeneration, enabling genomics-based comparisons of regeneration across species.

 

Increased serine synthesis provides an advantage for tumors arising in tissues where serine levels are limiting.

Cell Metab. 2019 Mar 13. pii: S1550-4131(19)30125-1. doi: 10.1016/j.cmet.2019.02.015.

Sullivan, M.R., Mattaini, K.R., Dennstedt, E.A., Nguyen, A.A., Sivanand, S., Reilly, M.F., Meeth, K., Muir, A., Darnell, A.M., Bosenberg, M.W., Lewis, C.A.*, and Vander Heiden, M.G.

Tumors exhibit altered metabolism compared to normal tissues. Many cancers upregulate expression of serine synthesis pathway enzymes, and some tumors exhibit copy-number gain of the gene encoding the first enzyme in the pathway, phosphoglycerate dehydrogenase (PHGDH). However, whether increased serine synthesis promotes tumor growth and how serine synthesis benefits tumors is controversial. Here, we demonstrate that increased PHGDH expression promotes tumor progression in mouse models of melanoma and breast cancer, human tumor types that exhibit PHGDH copy-number gain. We measure circulating serine levels and find that PHGDH expression is necessary to support cell proliferation at lower physiological serine concentrations. Increased dietary serine or high PHGDH expression is sufficient to increase intracellular serine levels and support faster tumor growth. Together, these data suggest that physiological serine availability restrains tumor growth and argue that tumors arising in serine-limited environments acquire a fitness advantage by upregulating serine synthesis pathway enzymes.

 

Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness.

Elife. 2019 Mar 7;8. pii: e43002. doi: 10.7554/eLife.43002.

Tye, B.W., Commins, N., Ryazanova, L.V., Wuhr, M., Springer, M., Pincus, D.*, and Churchman, L.S.

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

 

Amplification of a broad transcriptional program by a common factor triggers the meiotic cell cycle in mice.

Elife. 2019 Feb 27;8. pii: e43738. doi: 10.7554/eLife.43738.

Kojima, M.L.*, de Rooij, D.G.*, and Page, D.C.*

The germ line provides the cellular link between generations of multicellular organisms, its cells entering the meiotic cell cycle only once each generation. However, the mechanisms governing this initiation of meiosis remain poorly understood. Here, we examined cells undergoing meiotic initiation in mice, and we found that initiation involves the dramatic upregulation of a transcriptional network of thousands of genes whose expression is not limited to meiosis. This broad gene expression program is directly upregulated by STRA8, encoded by a germ cell-specific gene required for meiotic initiation. STRA8 binds its own promoter and those of thousands of other genes, including meiotic prophase genes, factors mediating DNA replication and the G1-S cell-cycle transition, and genes that promote the lengthy prophase unique to meiosis I. We conclude that, in mice, the robust amplification of this extraordinarily broad transcription program by a common factor triggers initiation of meiosis.

 

Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the Rag GTPases.

J Biol Chem. 2019 Feb 22;294(8):2970-2975. doi: 10.1074/jbc.AC119.007382.

Shen, K.*, Valenstein, M.L.*, Gu, X.*, and Sabatini, D.M.*

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex.

 

*Author affiliated with Whitehead Institute for Biomedical Research

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