Scientific Papers

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

Altered translation of GATA1 in Diamond-Blackfan anemia.

Nat Med. 2014 Jul;20(7):748-53.

Ludwig, L.S.*, Gazda, H.T., Eng, J.C.*, Eichhorn, S.W.*, Thiru, P.*, Ghazvinian, R., George, T.I., Gotlib, J.R., Beggs, A.H., Sieff, C.A., Lodish, H.F.*, Lander, E.S., and Sankaran, V.G.*

Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.

 

Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9.

PLoS One. 2014 Jun 27;9(6):e100450.

Sidik, S.M.*, Hackett, C.G.*, Tran, F., Westwood, N.J., and Lourido, S.*

Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.

 

Translation of small open reading frames within unannotated RNA transcripts in Saccharomyces cerevisiae.

Cell Rep. 2014 Jun 26;7(6):1858-66.

Smith, J.E., Alvarez-Dominguez, J.R., Kline, N., Huynh, N.J., Geisler, S., Hu, W.*, Coller, J., and Baker, K.E.

High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10-96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.

 

Piwi and potency: PIWI proteins in animal stem cells and regeneration.

Integr Comp Biol. 2014 Jun 19.

van Wolfswinkel, J.C.*

PIWI proteins are well known for their roles in the animal germline. They are essential for germline development and maintenance, and together with their binding partners, the piRNAs, they mediate transposon silencing. More recently, PIWI proteins have also been identified in somatic stem cells in diverse animals. The expression of PIWI proteins in these cells could be related to the ability of such cells to contribute to the germline. However, evaluation of stem cell systems across many different animal phyla suggests that PIWI proteins have an ancestral role in somatic stem cells, irrespective of their contribution to the germ cell lineage. Moreover, the data currently available reveal a possible correlation between the differentiation potential of a cell and its PIWI levels.

 

Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly.

J Immunol. 2014 Jun 15;192(12):6028-36.

Abdi, K., Singh, N.J., Spooner, E.*, Kessler, B.M., Radaev, S., Lantz, L., Xiao, T.S., Matzinger, P., Sun, P.D., and Ploegh, H.L.*

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.


Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells.

Stem Cell Reports. 2014 Jun 3;2(6):838-52.

Forster, R., Chiba, K., Schaeffer, L., Regalado, S.G., Lai, C.S., Gao, Q.*, Kiani, S., Farin, H.F., Clevers, H., Cost, G.J., Chan, A., Rebar, E.J., Urnov, F.D,, Gregory, P.D, Pachter, L., Jaenisch, R.*, and Hockemeyer, D.

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.

 

Genetic and chemical correction of cholesterol accumulation and impaired autophagy in hepatic and neural cells derived from Niemann-Pick type C patient-specific iPS cells.

Stem Cell Reports. 2014 May 15;2(6):866-80.

Maetzel, D.*, Sarkar, S.*, Wang, H., Abi-Mosleh, L., Xu, P., Cheng, A.W., Gao, Q.(, Mitalipova, M.*, and Jaenisch, R.*

Niemann-Pick type C (NPC) disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs) using transcription activator-like effector nucleases (TALENs). We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

 

The ADP-ribosyltransferase PARP10/ARTD10 interacts with proliferating cell nuclear antigen ( PCNA) and is required for DNA damage tolerance.

J Biol Chem. 2014 May 9;289(19):13627-37.

Nicolae, C.M., Aho, E.R., Vlahos, A.H.S., Choe, K.N., De, S., Karras, G.I.*, and Moldovan, G.L.

Background: PCNA mono-ubiquitination at stalled replication forks recruits translesion synthesis polymerases for fork restart. Results: The mono-ADP-ribosyltransferase PARP10 interacts with PCNA through a PIP-box. PARP10 knockdown results in DNA damage hypersensitivity and defective translesion synthesis. Conclusion: PARP10 participates in PCNA-dependent DNA damage tolerance. Significance: This is the first time that post-translational modification by mono-ADP-ribosylation is implicated in DNA repair. All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.

 

Cell-state transitions regulated by SLUG are critical for tissue regeneration and tumor initiation.

Stem Cell Reports. 2014 Apr 24;2(5):633-47.

Phillips, S., Prat, A., Sedic, M., Proia, T., Wronski, A., Mazumdar, S., Skibinski, A., Shirley, S.H., Perou, C.M., Gill, G., Gupta, P.B.*, and Kuperwasser, C.

Perturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis.

 

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development.

Haematologica. 2014 Apr;99(4):638-46.

Tsapogas, P., Swee, L.K.*, Nusser, A., Nuber, N., Kreuzaler, M., Capoferri, G., Rolink, H., Ceredig, R., and Rolink, A.

Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential.

 

 
*Author affiliated with Whitehead Institute for Biomedical Research

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