Scientific Papers

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

Self-organization and progenitor targeting generate stable patterns in planarian regeneration

Science. 2018 Mar 15.

Atabay, K.D.*, LoCascio, S.A.*, de Hoog, T.*, and Reddien, P.W.*

During animal regeneration cells must organize into discrete and functional systems. We show that self-organization, along with patterning cues, govern progenitor behavior in planarian regeneration. Surgical paradigms allowed manipulation of planarian eye regeneration in predictable locations and numbers, generating alternative stable neuroanatomical states for wild-type animals with multiple functional ectopic eyes. We utilized animals with multiple ectopic eyes and eye transplantation to demonstrate that broad progenitor specification, combined with self-organization, allows anatomy maintenance during regeneration. We propose a model for regenerative progenitors involving (i) migratory targeting cues, (ii) self-organization into existing or regenerating eyes, and (iii) a broad zone, associated with coarse progenitor specification, in which eyes can be targeted by progenitors. These three properties help explain how tissues can be organized during regeneration.


Withaferin A and Withanolide D analogues with dual heat-shock-inducing and cytotoxic activities: Semisynthesis and biological evaluation

J Nat Prod. 2018 Mar 14. 

Wijeratne, E.M.K., Oliveira, M.C.F., Mafezoli, J., Xu, Y.M., Minguzzi, S., Batista, P.H.J., Pessoa, O.D.L., Whitesell, L.*, and Gunatilaka, A.A.L.

Withanolides constitute a valuable class of bioactive natural products because some members of the class are known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA) and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations. Hitherto unknown 16beta-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine, providing unexpected 5beta-hydroxy-6alpha-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunninghamella echinulata and base-catalyzed cyclization of the resulting 16beta-hydroxy iodohydrins. Evaluation of these 25 withanolide analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure-activity relationships for WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA analogues. The 5beta,6beta-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16beta-OAc group to 4,27-di- O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4- O-acetyl-WD decreased both activities.


Yeast can accommodate phosphotyrosine: v-Src toxicity in yeast arises from a single disrupted pathway

FEMS Yeast Res. 2018 Mar 13.

Kritzer, J.A., Freyzon, Y.*, and Lindquist, S.*

Tyrosine phosphorylation is a key biochemical signal that controls growth and differentiation in multicellular organisms. Saccharomyces cerevisiae and nearly all other unicellular eukaryotes lack intact phosphotyrosine signaling pathways. However, many of these organisms have primitive phosphotyrosine-binding proteins and tyrosine phosphatases, leading to the assumption that the major barrier for emergence of phosphotyrosine signaling was the negative consequences of promiscuous tyrosine kinase activity. In this work, we reveal that the classic oncogene v-Src, which phosphorylates many dozens of proteins in yeast, is toxic because it disrupts a specific spore wall remodeling pathway. Using genetic selections, we find that expression of a specific cyclic peptide, or overexpression of SMK1, a MAP kinase that controls spore wall assembly, both lead to robust growth despite a continuous high level of phosphotyrosine in the yeast proteome. Thus, minimal genetic manipulations allow yeast to tolerate high levels of phosphotyrosine. These results indicate that the introduction of tyrosine kinases within single-celled organisms may not have been a major obstacle to the evolution of phosphotyrosine signaling.


BCL11B drives human mammary stem cell self-renewal in vitro by inhibiting basal differentiation

Stem Cell Reports. 2018 Mar 13;10(3):1131-1145.

Miller, D.H.*, Jin, D.X.*, Sokol, E.S.*, Cabrera, J.R., Superville, D.A.*, Gorelov, R.A.*, Kuperwasser, C., and Gupta, P.B.*

The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11B's functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues.


Dual role for inositol-requiring enzyme 1alpha in promoting the development of hepatocellular carcinoma during diet-induced obesity

Hepatology. 2018 Mar 5. 

Wu, Y., Shan, B., Dai, J., Xia, Z., Cai, J., Chen, T., Lv, S., Feng, Y.*, Zheng, L., Wang, Y., Liu, J., Fang, J., Xie, D., Rui, L., Liu, J., and Liu, Y.

Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1alpha (IRE1alpha), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1alpha in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in LKO mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and STAT3 activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1alpha abrogation blunted obesity-associated activation of hepatic IKKbeta-NF-kappaB pathway, leading to reduced production of the tumor-promoting inflammatory cytokines TNF and IL-6. Importantly, higher IRE1alpha expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: These results demonstrate that IRE1alpha acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. This article is protected by copyright. All rights reserved.


Translocon declogger Ste24 protects against IAPP oligomer-induced proteotoxicity

Cell. 2018 Mar 5.

Kayatekin, C.*, Amasino, A., Gaglia, G.*, Flannick, J., Bonner, J.M.*, Fanning, S.*, Narayan, P.*, Barrasa, M.I.*, Pincus, D.*, Landgraf, D.*, Nelson, J., Hesse, W.R., Costanzo, M.; AMP T2D-GENES Consortium, Myers, C.L., Boone, C., Florez, J.C., and Lindquist, S.*

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to beta cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to beta cell failure.


MiR221 promotes precursor B-cell retention in the bone marrow by amplifying the PI3K-signaling pathway in mice

Eur J Immunol. 2018 Mar 5. 

Petkau, G., Kawano, Y., Wolf, I., Knoll, M.*, and Melchers, F. 

Hematopoietic stem cells and lineage-uncommitted progenitors are able to home to the bone marrow upon transplantation and reconstitute the host with hematopoietic progeny. Expression of miR221 in B-lineage committed preBI-cells induces their capacity to home to the bone marrow. However, the molecular mechanisms underlying miR221-controlled bone marrow homing and retention remain poorly understood. Here, we demonstrate, that miR221 regulates bone marrow retention of such B-cell precursors by targeting PTEN, thus enhancing PI3K signaling in response to the chemokine CXCL12. MiR221-enhanced PI3K signaling leads to increased expression of the anti-apoptotic protein Bcl2 and VLA4 integrin-mediated adhesion to VCAM1 in response to CXCL12 in vitro. Ablation of elevated PI3K activity abolishes the retention of miR221 expressing preBI-cells in the bone marrow. These results suggest that amplification of PI3K signaling by miR221 could be a general mechanism for bone marrow residence, shared by miR221-expressing hematopoietic cells.


Quantitative analysis of population-scale family trees with millions of relatives

Science. 2018 Mar 1. 

Kaplanis, J.*, Gordon, A.*, Shor, T., Weissbrod, O., Geiger, D., Wahl, M.*, Gershovits, M.*, Markus, B.*, Sheikh, M.*, Gymrek, M.*, Bhatia, G., MacArthur, D.G., Price, A.L., and Erlich, Y.*

Family trees have vast applications in multiple fields from genetics to anthropology and economics. However, the collection of extended family trees is tedious and usually relies on resources with limited geographical scope and complex data usage restrictions. Here, we collected 86 million profiles from publicly-available online data shared by genealogy enthusiasts. After extensive cleaning and validation, we obtained population-scale family trees, including a single pedigree of 13 million individuals. We leveraged the data to partition the genetic architecture of longevity by inspecting millions of relative pairs and to provide insights into the geographical dispersion of families. We also report a simple digital procedure to overlay other datasets with our resource in order to empower studies with population-scale genealogical data.


Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition

Elife. 2018 Feb 26;7.

Hara, M.*, Lourido, S.*, Petrova, B.*, Lou, H.J., Von Stetina, J.R.*, Kashevsky, H.*, Turk, B.E., and Orr-Weaver, T.L.*

The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.


Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

Nat Protoc. 2017 Oct;12(10):2215-2231. 

Chen, W.W.*, Freinkman, E.*, and Sabatini, D.M.*

Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in approximately 12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.


*Author affiliated with Whitehead Institute for Biomedical Research

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