Scientific Papers

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

"Donating our bodies to science": A discussion about autopsy and organ donation in Turner syndrome.

Am J Med Genet C Semin Med Genet. 2019 Jan 11. doi: 10.1002/ajmg.c.31671.

Prakash, S.K., San Roman, A.K.*, Crenshaw, M., Flink, B., Earle, K., Los, E., Bonnard, A., and Lin, A.E.

At the Third Turner Resource Network Symposium, a working group presented the results of collaborative discussions about the importance of autopsy in Turner syndrome (TS). Considerable gaps in understanding the causes of death in TS can only be closed by more frequent death investigations and autopsies. The presentation included an overview of autopsy methods, strategies for utilizing autopsy, and biobanking to address research questions about TS, and the role of palliative care in the context of autopsy. This review highlights strategies to promote autopsy and tissue donation, culminating with an action plan to increase autopsy rates in the TS community.

 

Reducing phenolic off-flavors through CRISPR-based gene editing of the FDC1 gene in Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid lager beer yeasts.

PLoS One. 2019 Jan 9;14(1):e0209124. doi: 10.1371/journal.pone.0209124.

Mertens, S., Gallone, B., Steensels, J., Herrera-Malaver, B., Cortebeek, J., Nolmans, R., Saels, V., Vyas, V.K.*, and Verstrepen, K.J.

Today's beer market is challenged by a decreasing consumption of traditional beer styles and an increasing consumption of specialty beers. In particular, lager-type beers (pilsner), characterized by their refreshing and unique aroma and taste, yet very uniform, struggle with their sales. The development of novel variants of the common lager yeast, the interspecific hybrid Saccharomyces pastorianus, has been proposed as a possible solution to address the need of product diversification in lager beers. Previous efforts to generate new lager yeasts through hybridization of the ancestral parental species (S. cerevisiae and S. eubayanus) yielded strains with an aromatic profile distinct from the natural biodiversity. Unfortunately, next to the desired properties, these novel yeasts also inherited unwanted characteristics. Most notably is their phenolic off-flavor (POF) production, which hampers their direct application in the industrial production processes. Here, we describe a CRISPR-based gene editing strategy that allows the systematic and meticulous introduction of a natural occurring mutation in the FDC1 gene of genetically complex industrial S. cerevisiae strains, S. eubayanus yeasts and interspecific hybrids. The resulting cisgenic POF- variants show great potential for industrial application and diversifying the current lager beer portfolio. 

 

Ectopic activation of the spindle assembly checkpoint signaling cascade reveals its biochemical design.

Curr Biol. 2019 Jan 7;29(1):104-119.e10. doi: 10.1016/j.cub.2018.11.054.

Chen, C.*, Whitney, I.P.*, Banerjee, A., Sacristan, C., Sekhri, P., Kern, D.M.*, Fontan, A., Kops, G., Tyson, J.J., Cheeseman, I.M.*, and Joglekar AP.

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

  

Phenotypic plasticity: driver of cancer initiation, progression, and therapy resistance.

Cell Stem Cell. 2019 Jan 3;24(1):65-78. doi: 10.1016/j.stem.2018.11.011. 

Gupta, P.B.*, Pastushenko, I., Skibinski, A., Blanpain, C., and Kuperwasser, C.

Our traditional understanding of phenotypic plasticity in adult somatic cells comprises dedifferentiation and transdifferentiation in the context of tissue regeneration or wound healing. Although dedifferentiation is central to tissue repair and stemness, this process inherently carries the risk of cancer initiation. Consequently, recent research suggests phenotypic plasticity as a new paradigm for understanding cancer initiation, progression, and resistance to therapy. Here, we discuss how cells acquire plasticity and the role of plasticity in initiating cancer, cancer progression, and metastasis and in developing therapy resistance. We also highlight the epithelial-to-mesenchymal transition (EMT) and known molecular mechanisms underlying plasticity and we consider potential therapeutic avenues. 

 

Heat shock factor 1 drives intergenic association of its target gene loci upon heat shock.

Cell Rep. 2019 Jan 2;26(1):18-28.e5. doi: 10.1016/j.celrep.2018.12.034.

Chowdhary, S., Kainth, A.S., Pincus, D.*, and Gross, D.S.

Transcriptional induction of heat shock protein (HSP) genes is accompanied by dynamic changes in their 3D structure and spatial organization, yet the molecular basis for these phenomena remains unknown. Using chromosome conformation capture and single-cell imaging, we show that genes transcriptionally activated by Hsf1 specifically interact across chromosomes and coalesce into diffraction-limited intranuclear foci. Genes activated by the alternative stress regulators Msn2/Msn4, in contrast, do not interact among themselves nor with Hsf1 targets. Likewise, constitutively expressed genes, even those interposed between HSP genes, show no detectable interaction. Hsf1 forms discrete subnuclear puncta when stress activated, and these puncta dissolve in concert with transcriptional attenuation, paralleling the kinetics of HSP gene coalescence and dissolution. Nuclear Hsf1 and RNA Pol II are both necessary for intergenic HSP gene interactions, while DNA-bound Hsf1 is necessary and sufficient to drive heterologous gene coalescence. Our findings demonstrate that Hsf1 can dynamically restructure the yeast genome.

 

Editorial: In memory of Susan Lindquist.

FEMS Yeast Res. 2019 Jan 1;19(1). doi: 10.1093/femsyr/foy110.

Bevis, B.J.*

  

The molecular structure of plant sporopollenin.

Nat Plants. 2019 Jan;5(1):41-46. doi: 10.1038/s41477-018-0330-7. 

Li, F.S.*, Phyo, P., Jacobowitz, J.*, Hong, M., and Weng, J.K.*

Sporopollenin is a ubiquitous and extremely chemically inert biopolymer that constitutes the outer wall of all land-plant spores and pollen grains(1). Sporopollenin protects the vulnerable plant gametes against a wide range of environmental assaults, and is considered a prerequisite for the migration of early plants onto land(2). Despite its importance, the chemical structure of plant sporopollenin has remained elusive(1). Using a newly developed thioacidolysis degradative method together with state-of-the-art solid-state NMR techniques, we determined the detailed molecular structure of pine sporopollenin. We show that pine sporopollenin is primarily composed of aliphatic-polyketide-derived polyvinyl alcohol units and 7-O-p-coumaroylated C16 aliphatic units, crosslinked through a distinctive dioxane moiety featuring an acetal. Naringenin was also identified as a minor component of pine sporopollenin. This discovery answers the long-standing question about the chemical make-up of plant sporopollenin, laying the foundation for future investigations of sporopollenin biosynthesis and for the design of new biomimetic polymers with desirable inert properties.

 

Genome-wide CRISPR/Cas9 screening for identification of cancer genes in cell lines.

Methods Mol Biol. 2019;1907:125-136. doi: 10.1007/978-1-4939-8967-6_10.

Adelmann, C.H., Wang, T., Sabatini, D.M.*, and Lander, E.S.

In this protocol, pooled sgRNA libraries targeting thousands of genes are computationally designed, generated using microarray-based synthesis techniques, and packaged into lentiviral particles. Target cells of interest are transduced with the lentiviral sgRNA pools to generate a collection of knockout mutants-via Cas9-mediated genomic cleavage-and screened for a phenotype of interest. The relative abundance of each mutant in the population can be monitored over time through high-throughput sequencing of the integrated sgRNA expression cassettes. Using this technique, we outline strategies for the identification of cancer driver genes and genes mediating drug response.

 

Non-overlapping control of transcriptome by promoter- and super-enhancer-associated dependencies in multiple myeloma.

Cell Rep. 2018 Dec 26;25(13):3693-3705.e6. doi: 10.1016/j.celrep.2018.12.016.

Fulciniti, M., Lin, C.Y., Samur, M.K., Lopez, M.A., Singh, I., Lawlor, M.A., Szalat, R.E., Ott, C.J., Avet-Loiseau, H., Anderson, K.C., Young, R.A.*, Bradner, J.E., and Munshi, N.C.

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy.

 

One thousand simple rules.

PLoS Comput Biol. 2018 Dec 20;14(12):e1006670. doi: 10.1371/journal.pcbi.1006670. 

Bourne, P.E., Lewitter, F.*, Markel, S., and Papin, J.A.

  

Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles.

Biomater Sci. 2018 Dec 18;7(1):113-124. doi: 10.1039/c8bm01208c.

Yang, Y.S.S., Moynihan, K.D., Bekdemir, A., Dichwalkar, T.M., Noh, M.M., Watson, N.*, Melo, M., Ingram, J.*, Suh, H., Ploegh, H.*, Stellacci, F.R., and Irvine, D.J.

We sought to develop a nanoparticle vehicle that could efficiently deliver small molecule drugs to target lymphocyte populations. The synthesized amphiphilic organic ligand-protected gold nanoparticles (amph-NPs) were capable of sequestering large payloads of small molecule drugs within hydrophobic pockets of their ligand shells. These particles exhibit membrane-penetrating activity in mammalian cells, and thus enhanced uptake of a small molecule TGF-inhibitor in T cells in cell culture. By conjugating amph-NPs with targeting antibodies or camelid-derived nanobodies, the particles' cell-penetrating properties could be temporarily suppressed, allowing targeted uptake in specific lymphocyte subpopulations. Degradation of the protein targeting moieties following particle endocytosis allowed the NPs to recover their cell-penetrating activity in situ to enter the cytoplasm of T cells. In vivo, targeted amph-NPs showed 40-fold enhanced uptake in CD8(+) T cells relative to untargeted particles, and delivery of TGF- inhibitor-loaded particles to T cells enhanced their cytokine polyfunctionality in a cancer vaccine model. Thus, this system provides a facile approach to concentrate small molecule compounds in target lymphocyte populations of interest for immunotherapy in cancer and other diseases.

  

RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway.

Sci Adv. 2018 Nov 21;4(11):eaav0443. doi: 10.1126/sciadv.aav0443. 

Heo, J.M., Ordureau, A., Swarup, S., Paulo, J.A., Shen, K.*, Sabatini, D.M.*, and Harper, J.W.

Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7A(S72), but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7A(S72E) with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7A(S72A). Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.

 

 

*Author affiliated with Whitehead Institute for Biomedical Research

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