Scientific Papers

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

mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues.

Mol Cell. 2014 Oct 2;56(1):104-15.

Eichhorn, S.W.*8, Guo, H.*, McGeary, S.E.*, Rodriguez-Mias, R.A., Shin, C.*, Baek, D.*, Hsu, S.H., Ghoshal, K., Villen, J., and Bartel, D.P.*

MicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined globally. Here, we extend the global steady-state measurements to additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition, or translational state, mRNA destabilization explains most (66%->90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results imply that consequential miRNA-mediated repression is largely irreversible and provide other insights into the nature of miRNA-mediated regulation. They also simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects.

Negative self-regulation of TLR9 signaling by its N-terminal proteolytic cleavage product.

J Immunol. 2014 Oct 1;193(7):3726-35.

Lee, S., Kang, D., Ra, E.A., Lee, T.A., Ploegh, H.L.*, and Park, B.

TLR signaling is essential to innate immunity against microbial invaders and must be tightly controlled. We have previously shown that TLR9 undergoes proteolytic cleavage processing by lysosomal proteases to generate two distinct fragments. The C-terminal cleavage product plays a critical role in activating TLR9 signaling; however, the precise role of the N-terminal fragment, which remains in lysosomes, in the TLR9 response is still unclear. In this article, we report that the N-terminal cleavage product negatively regulates TLR9 signaling. Notably, the N-terminal fragment promotes the aspartic protease-mediated degradation of the C-terminal fragment in endolysosomes. Furthermore, the N-terminal TLR9 fragment physically interacts with the C-terminal product, thereby inhibiting the formation of homodimers of the C-terminal fragment; this suggests that the monomeric C-terminal product is more susceptible to attack by aspartic proteases. Together, these results suggest that the N-terminal TLR9 proteolytic cleavage product is a negative self-regulator that prevents excessive TLR9 signaling activity.

Poised chromatin in the mammalian germ line.

Development. 2014 Oct;141(19):3619-26.

Lesch, B.J.*, and Page, D.C.*

Poised (bivalent) chromatin is defined by the simultaneous presence of histone modifications associated with both gene activation and repression. This epigenetic feature was first observed at promoters of lineage-specific regulatory genes in embryonic stem cells in culture. More recent work has shown that, in vivo, mammalian germ cells maintain poised chromatin at promoters of many genes that regulate somatic development, and that they retain this state from fetal stages through meiosis and gametogenesis. We hypothesize that the poised chromatin state is essential for germ cell identity and function. We propose three roles for poised chromatin in the mammalian germ line: prevention of DNA methylation, maintenance of germ cell identity and preparation for totipotency. We discuss these roles in the context of recently proposed models for germline potency and epigenetic inheritance.

Cpeb4-mediated translational regulatory circuitry controls terminal erythroid differentiation.

Dev Cell. 2014 Sep 29;30(6):660-72.

Hu, W.*, Yuan, B.*, and Lodish, H.F.*

While we have considerable understanding of the transcriptional networks controlling mammalian cell differentiation, our knowledge of posttranscriptional regulatory events is very limited. Using differentiation of primary erythroid cells as a model, we show that the sequence-specific mRNA-binding protein Cpeb4 is strongly induced by the erythroid-important transcription factors Gata1 and Tal1 and is essential for terminal erythropoiesis. By interacting with the translation initiation factor eIF3, Cpeb4 represses the translation of a large set of mRNAs, including its own mRNA. Thus, transcriptional induction and translational repression combine to form a negative feedback loop to control Cpeb4 protein levels within a specific range that is required for terminal erythropoiesis. Our study provides an example of how translational control is integrated with transcriptional regulation to precisely control gene expression during mammalian cell differentiation.

Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

Cell. 2014 Sep 25;159(1):148-62.

Schwartz, S., Bernstein, D.A., Mumbach, M.R., Jovanovic, M., Herbst, R.H., Leon-Ricardo, B.X., Engreitz, J.M., Guttman, M., Satija, R., Lander, E.S., Fink, G.*, and Regev, A.

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Psi-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Psi-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.

The Sestrins interact with GATOR2 to negatively regulate the amino-acid-sensing pathway upstream of mTORC1.

Cell Rep. 2014 Sep 24.

Chantranupong, L.*, Wolfson, R.L.*, Orozco, J.M.*, Saxton, R.A.*, Scaria, S.M.*, Bar-Peled, L.*, Spooner, E.*, Isasa, M., Gygi, S.P., and Sabatini, D.M.*

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a major regulator of cell growth that responds to numerous environmental cues. A key input is amino acids, which act through the heterodimeric Rag GTPases (RagA or RagB bound to RagC or RagD) in order to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. GATOR2 is a complex of unknown function that positively regulates mTORC1 signaling by acting upstream of or in parallel to GATOR1, which is a GTPase-activating protein (GAP) for RagA or RagB and an inhibitor of the amino-acid-sensing pathway. Here, we find that the Sestrins, a family of poorly understood growth regulators (Sestrin1-Sestrin3), interact with GATOR2 in an amino-acid-sensitive fashion. Sestrin2-mediated inhibition of mTORC1 signaling requires GATOR1 and the Rag GTPases, and the Sestrins regulate the localization of mTORC1 in response to amino acids. Thus, we identify the Sestrins as GATOR2-interacting proteins that regulate the amino-acid-sensing branch of the mTORC1 pathway.

De-differentiation confers multidrug resistance via noncanonical PERK-Nrf2 signaling.

PLoS Biol. 2014 Sep 9;12(9):e1001945.

Del Vecchio, C.A.*, Feng, Y.*, Sokol, E.S., Tillman, E.J., Sanduja, S.*, Reinhardt, F.*, and Gupta, P.B.*

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy.

A critical role for mTORC1 in erythropoiesis and anemia.

Elife. 2014 Sep 8;3.

Knight, Z.A., Schmidt, S.F., Birsoy, K.*, Tan, K., and Friedman, J.M.

Red blood cells (RBC) must coordinate their rate of growth and proliferation with the availability of nutrients, such as iron, but the signaling mechanisms that link nutritional state to RBC growth are incompletely understood. We performed a screen for cell types that have high levels of signaling through mTORC1, a protein kinase that couples nutrient availability to cell growth. This screen revealed that reticulocytes show high levels of phosphorylated ribosomal protein S6, a downstream target of mTORC1. We found that mTORC1 activity in RBCs is regulated by dietary iron, and that genetic activation or inhibition of mTORC1 results in macrocytic or microcytic anemia, respectively. Finally, ATP competitive mTOR inhibitors reduced RBC proliferation and were lethal after treatment with phenylhydrazine, an inducer of hemolysis. These results identify the mTORC1 pathway as a critical regulator of RBC growth and proliferation, and establish that perturbations in this pathway result in anemia.

The developmental potential of iPSCs is greatly influenced by reprogramming factor selection.

Cell Stem Cell. 2014 Sep 4;15(3):295-309.

Buganim, Y., Markoulaki, S.*, van Wietmarschen, N., Hoke, H.*, Wu, T., Ganz, K.*, Akhtar-Zaidi, B.*, He, Y.*, Abraham, B.J., Porubsky, D.*, Kulenkampff, E.*, Faddah, D.A.*, Shi, L.*, Gao, Q.*, Sarkar, S.*, Cohen, M.*, Goldmann, J.*, Nery, J.R., Schultz, M.D., Ecker, J.R., Xiao, A., Young, R.A.*, Lansdorp ,P.M., and Jaenisch, R.*

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

SirT1 is required in the male germ cell for differentiation and fecundity in mice.

Development. 2014 Sep;141(18):3495-504.

Bell, E.L., Nagamori, I., Williams, E.O., Del Rosario, A.M., Bryson, B.D., Watson, N.*, White, F.M., Sassone-Corsi, P., and Guarente, L.

Sirtuins are NAD(+)-dependent deacylases that regulate numerous biological processes in response to the environment. SirT1 is the mammalian ortholog of yeast Sir2, and is involved in many metabolic pathways in somatic tissues. Whole body deletion of SirT1 alters reproductive function in oocytes and the testes, in part caused by defects in central neuro-endocrine control. To study the function of SirT1 specifically in the male germ line, we deleted this sirtuin in male germ cells and found that mutant mice had smaller testes, a delay in differentiation of pre-meiotic germ cells, decreased spermatozoa number, an increased proportion of abnormal spermatozoa and reduced fertility. At the molecular level, mutants do not have the characteristic increase in acetylation of histone H4 at residues K5, K8 and K12 during spermiogenesis and demonstrate corresponding defects in the histone to protamine transition. Our findings thus reveal a germ cell-autonomous role of SirT1 in spermatogenesis.

*Author affiliated with Whitehead Institute for Biomedical Research

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