Cell Reports
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Aug 21, 2014

Volume 8Issue 4p923-1240
Open Access
On the cover: During brain development, the neurons that connect the eyes to the brain (RGCs) direct their axons to specific brain targets, a process called axon-target matching. In this issue, Osterhout et al. show that different RGC types match to their targets with different strategies: some make a lot of errors and correct them, whereas others make very few. The strategy used correlates with birthdate with early-born RGCs making the most errors. The cover depicts clocks linked together as gears as a metaphor of how timing of neuron birth may generally impact development of neural circuit connections. Original image: Adam Gault....
On the cover: During brain development, the neurons that connect the eyes to the brain (RGCs) direct their axons to specific brain targets, a process called axon-target matching. In this issue, Osterhout et al. show that different RGC types match to their targets with different strategies: some make a lot of errors and correct them, whereas others make very few. The strategy used correlates with birthdate with early-born RGCs making the most errors. The cover depicts clocks linked together as gears as a metaphor of how timing of neuron birth may generally impact development of neural circuit connections. Original image: Adam Gault.


  • Sex-Dependent Gene Expression in Human Pluripotent Stem Cells

    • Daniel Ronen,
    • Nissim Benvenisty
    Sexually dimorphic traits are mostly hormonally driven and are established following gonadal development. By focusing on Y chromosome transcription in undifferentiated human embryonic stem cells, Ronen and Benvenisty now show that the male-determining gene SRY is expressed in these cells. This transcription factor, along with its target autosomal genes, may drive early differences in steroid metabolism and neuronal development between male and female embryos.
  • Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury

    • Johanna R. Schaub,
    • Yann Malato,
    • Coralie Gormond,
    • Holger Willenbring
    Schaub et al. test the long-standing belief that liver stem cells give rise to new hepatocytes in the chronically injured liver to compensate for impaired hepatocyte proliferation. They fail to find evidence for such a backup system for hepatocyte regeneration in a common mouse model of chronic liver injury.
  • Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

    • Ann-Marie Baker,
    • Biancastella Cereser,
    • Samuel Melton,
    • Alexander G. Fletcher,
    • Manuel Rodriguez-Justo,
    • Paul J. Tadrous,
    • Adam Humphries,
    • George Elia,
    • Stuart A.C. McDonald,
    • Nicholas A. Wright,
    • Benjamin D. Simons,
    • Marnix Jansen,
    • Trevor A. Graham
    Baker et al. examine the in vivo stem cell biology of human colonic crypts. They reveal that each crypt contains a small number of functional stem cells and that stem cell division is predominantly symmetric and also quantify perturbation of stem cell architecture within adenomas. Additionally, they measure the division rate of human colonic crypts as once every 30–40 years in the healthy colon and demonstrate that the crypt division rate is increased 10-fold within small adenomas.
  • Balanced Activity of Three Mitotic Motors Is Required for Bipolar Spindle Assembly and Chromosome Segregation

    • Roy G.H.P. van Heesbeen,
    • Marvin E. Tanenbaum,
    • René H. Medema
    The bipolar spindle is a microtubule network required for faithful segregation of chromosomes during mitosis. The assembly of the bipolar spindle depends on forces generated by microtubule motor proteins. Van Heesbeen et al. now show how three microtubule motor proteins act together to produce the right force balance in order to build a robust bipolar spindle. They reveal that force imbalances result in defects in spindle architecture and alignment of the chromosomes.
  • Stabilization of Cartwheel-less Centrioles for Duplication Requires CEP295-Mediated Centriole-to-Centrosome Conversion

    • Denisse Izquierdo,
    • Won-Jing Wang,
    • Kunihiro Uryu,
    • Meng-Fu Bryan Tsou
    Vertebrate centrioles naturally lose their geometric scaffold, the cartwheel, during mitosis, before they support duplication in the following S phase. Whether these “cartwheel-less” centrioles are intrinsically stable or must be maintained through other modifications remains unclear. Izquierdo et al. now find that cartwheel removal can have grave repercussions for centriole stability and that centriole-to-centrosome conversion mediated by CEP295 must occur in parallel during mitosis to maintain cartwheel-less centrioles for duplication in the S phase.
  • A Pseudouridine Residue in the Spliceosome Core Is Part of the Filamentous Growth Program in Yeast

    • Anindita Basak,
    • Charles C. Query
    Pseudouridines (Ψ) are C5-glycoside isomers of uridine. Although they are implicated in enhanced local RNA stacking or altered protein-RNA interactions, their physiologic effects are mostly unknown. Here, Basak and Query report a Pus1p-dependent pseudouridine residue (U6-Ψ28) that is induced in the Saccharomyces cerevisiae spliceosome during filamentous growth, a morphogenic switch that occurs in response to environmental stress. They show that Pus1p-dependent pseudouridylation of U6 snRNA contributes to the filamentation growth program.
  • Single-Cell Imaging of Caspase-1 Dynamics Reveals an All-or-None Inflammasome Signaling Response

    • Ting Liu,
    • Yoshifumi Yamaguchi,
    • Yoshitaka Shirasaki,
    • Koichi Shikada,
    • Mai Yamagishi,
    • Katsuaki Hoshino,
    • Tsuneyasu Kaisho,
    • Kiwamu Takemoto,
    • Toshihiko Suzuki,
    • Erina Kuranaga,
    • Osamu Ohara,
    • Masayuki Miura
    Inflammasomes are key regulators of innate immunity through caspase-1 activation that induces proinflammatory cytokine production and cell death. How inflammasomes and caspase-1 activation occurs at the single-cell level is largely unknown. Using a genetically encoded FRET sensor to monitor caspase-1 activity in a single macrophage, Liu et al. find that caspase-1 is activated in an all-or-none manner, irrespective of the type or strength of stimuli. This activation induces the digital secretion of a proinflammatory cytokine independent of cell death.
  • A Polymorphic Enhancer near GREM1 Influences Bowel Cancer Risk through Differential CDX2 and TCF7L2 Binding

    • Annabelle Lewis,
    • Luke Freeman-Mills,
    • Elisa de la Calle-Mustienes,
    • Rosa María Giráldez-Pérez,
    • Hayley Davis,
    • Emma Jaeger,
    • Martin Becker,
    • Nina C. Hubner,
    • Luan N. Nguyen,
    • Jorge Zeron-Medina,
    • Gareth Bond,
    • Hendrik G. Stunnenberg,
    • Jaime J. Carvajal,
    • Jose Luis Gomez-Skarmeta,
    • Simon Leedham,
    • Ian Tomlinson
    Lewis et al. investigate how a common polymorphism, rs16969681, near the GREM1 gene predisposes to bowel cancer. They show that rs16969681 lies at an enhancer of GREM1 mRNA expression. The rs16969681 alleles differentially bind the intestine-specific transcription factor CDX2 and the Wnt effector TCF7L2, with the cancer risk allele showing stronger binding. This results in differences in GREM1 expression among individuals with the three rs16969681 genotypes. Different levels of GREM1 modulate the growth of colorectal tumors.
  • Perturbation of NCOA6 Leads to Dilated Cardiomyopathy

    • Jae-il Roh,
    • Cheolho Cheong,
    • Young Hoon Sung,
    • Jeehyun Lee,
    • Jaewon Oh,
    • Beom Seob Lee,
    • Jong-Eun Lee,
    • Yong Song Gho,
    • Duk-Kyung Kim,
    • Chan Bae Park,
    • Ji Hyun Lee,
    • Jae Woon Lee,
    • Seok-Min Kang,
    • Han-Woong Lee
    Dilated cardiomyopathy (DCM) is a type of heart muscle disease and one cause of congestive heart failure. Because approximately 40% of DCM patients exhibit evidence of autosomal inheritance, the search for susceptibility loci is a major challenge in DCM research. Here, Roh et al. show that depletion of NCOA6 leads to DCM accompanied by impaired mitochondrial function and reduced peroxisome proliferator-activated receptor δ activity. NCOA6 genetic mutations found in patients with DCM suggest a role in normal heart function.
  • BRCA1 Is a Histone-H2A-Specific Ubiquitin Ligase

    • Reinhard Kalb,
    • Donna L. Mallery,
    • Conor Larkin,
    • Jeffrey T.J. Huang,
    • Kevin Hiom
    The ubiquitin ligase (E3) activity of BRCA1 is its only known biochemical activity in vitro. Kalb et al. report that the heterodimeric complex formed by BRCA1/BARD1 ubiquitylates histone H2A in nucleosomes specifically at its C-terminal tail. Moreover, in vitro and in vivo assays identify H2A lysines127 and 129 as the target lysines. These results help to explain the localization and activity of BRCA1/BARD1 on chromatin in cells.
  • Birthdate and Outgrowth Timing Predict Cellular Mechanisms of Axon Target Matching in the Developing Visual Pathway

    • Jessica A. Osterhout,
    • Rana N. El-Danaf,
    • Phong L. Nguyen,
    • Andrew D. Huberman
    How neurons recognize their correct targets during mammalian brain development remains unclear. In this study, Osterhout et al. explore this question in the developing visual system using mice where the neurons that connect the eyes to the brain are fluorescent. In fact, these neurons use widely varying strategies to select their targets; some sample a lot of targets and then correct errors, whereas others make accurate choices from the outset. The degree of error making and correction appears closely related to when a neuron is born. These findings help explain how complex brain circuits are built.
  • Sox6 and Otx2 Control the Specification of Substantia Nigra and Ventral Tegmental Area Dopamine Neurons

    • Lia Panman,
    • Maria Papathanou,
    • Ariadna Laguna,
    • Tony Oosterveen,
    • Nikolaos Volakakis,
    • Dario Acampora,
    • Idha Kurtsdotter,
    • Takashi Yoshitake,
    • Jan Kehr,
    • Eliza Joodmardi,
    • Jonas Muhr,
    • Antonio Simeone,
    • Johan Ericson,
    • Thomas Perlmann
    Panman et al. provide detailed insight into how distinct subtypes of dopamine neurons are generated, including those that are particularly affected in Parkinson’s disease. They show that the transcription factor Sox6 promotes the generation of the most vulnerable type of dopamine neurons in the substantia nigra. Expression of Sox6 is also shown to be diminished in Parkinson’s disease. This knowledge should prove important for the development of stem cell engineering of dopamine neurons for cell replacement therapy.


  • Costs and Benefits of Mutational Robustness in RNA Viruses

    • Adi Stern,
    • Simone Bianco,
    • Ming Te Yeh,
    • Caroline Wright,
    • Kristin Butcher,
    • Chao Tang,
    • Rasmus Nielsen,
    • Raul Andino
    RNA viruses mutate rapidly and must adapt to multiple diverse environments. It has been suggested that viruses buffer the effects of deleterious mutations via mechanisms of mutational robustness. This allows populations to accumulate neutral diversity and use this diversity to adapt to new challenges. Using a combined theoretical and experimental approach, Andino and colleagues show that neutral diversity can also be harmful for the virus in the new environment. This study challenges the notion that mutational robustness is always beneficial for virus populations.
  • Oncogene Mimicry as a Mechanism of Primary Resistance to BRAF Inhibitors

    • Martin L. Sos,
    • Rebecca S. Levin,
    • John D. Gordan,
    • Juan A. Oses-Prieto,
    • James T. Webber,
    • Megan Salt,
    • Byron Hann,
    • Alma L. Burlingame,
    • Frank McCormick,
    • Sourav Bandyopadhyay,
    • Kevan M. Shokat
    The treatment of BRAF-mutant tumors has been revolutionized by the development of RAF inhibitors. However, a substantial part of tumors display mechanisms that render these drugs ineffective. Sos et al. use a chemical proteomic approach to systematically characterize these escape mechanisms in BRAF-mutant cell lines. The authors identify components of the RAS-RAF pathway as well as autocrine IL-6 secretion as unexpected sources of resistance signaling in BRAF-mutant cancer.
  • A Macrohistone Variant Links Dynamic Chromatin Compaction to BRCA1-Dependent Genome Maintenance

    • Simran Khurana,
    • Michael J. Kruhlak,
    • Jeongkyu Kim,
    • Andy D. Tran,
    • Jinping Liu,
    • Katherine Nyswaner,
    • Lei Shi,
    • Parthav Jailwala,
    • Myong-Hee Sung,
    • Ofir Hakim,
    • Philipp Oberdoerffer
    Accurate DNA double-strand break (DSB) repair relies on the choice between two central but opposing repair effectors, BRCA1 and 53BP1, which occupy large, DSB-flanking chromatin domains. The mechanisms that regulate this process remain largely unexplored. Khurana et al. now show that the macrohistone variant macroH2A1 and the H3K9 methyltransferase PRDM2 link the dynamic formation of repressive chromatin at DSBs to selective BRCA1 recruitment, homology-directed repair, and BRCA1-dependent genome maintenance.
  • Escape from Telomere-Driven Crisis Is DNA Ligase III Dependent

    • Rhiannon E. Jones,
    • Sehyun Oh,
    • Julia W. Grimstead,
    • Jacob Zimbric,
    • Laureline Roger,
    • Nicole H. Heppel,
    • Kevin E. Ashelford,
    • Kate Liddiard,
    • Eric A. Hendrickson,
    • Duncan M. Baird
    Escape from crisis caused by telomere fusion is a fundamental process that drives genomic instability, clonal evolution, and malignant progression in human cells. Jones et al. show that a specific form of telomere fusion mediated by ligase III but not ligase IV is required for human cells to escape crisis. They also reveal distinct mutational profiles that accompany fusion mediated by ligases III or IV. They consider that this profile is important in determining the ability of cells to survive a telomere-driven crisis.
  • Nontranscriptional Role of Hif-1α in Activation of γ-Secretase and Notch Signaling in Breast Cancer

    • Jennifer C. Villa,
    • Danica Chiu,
    • Alissa H. Brandes,
    • Freddy E. Escorcia,
    • Carlos H. Villa,
    • William F. Maguire,
    • Cheng-Jun Hu,
    • Elisa de Stanchina,
    • M. Celeste Simon,
    • Sangram S. Sisodia,
    • David A. Scheinberg,
    • Yue-Ming Li
    Villa et al. offer a mechanism by which γ-secretase is temporally regulated through a role of Hif-1α independent of its function as a transcriptional regulator during hypoxia, and demonstrate that γ-secretase displays enzymatic plasticity in response to hypoxia through a direct interaction with Hif-1α.
  • Gli3 Repressor Controls Cell Fates and Cell Adhesion for Proper Establishment of Neurogenic Niche

    • Hui Wang,
    • Anna W. Kane,
    • Cheol Lee,
    • Sohyun Ahn
    Neurons are continuously produced from the neurogenic niche in the mature brain, but little is known about how the subventricular zone (SVZ) niche forms. Ahn and colleagues find that Gli3 repressor (Gli3R), a repressor of Sonic Hedgehog signaling, plays a critical role during SVZ niche establishment. They provide molecular evidence that Gli3R regulates ependymal cell fate specification through suppression of gp130/STAT3 signaling. Furthermore, they demonstrate that Gli3R regulates cell adhesion and organization of the niche via maintenance of Numb.
  • Organization of Monosynaptic Inputs to the Serotonin and Dopamine Neuromodulatory Systems

    • Sachie K. Ogawa,
    • Jeremiah Y. Cohen,
    • Dabin Hwang,
    • Naoshige Uchida,
    • Mitsuko Watabe-Uchida
    Serotonin or dopamine neurons project throughout the brain and control many behaviors. Despite their importance, little is known about how serotonin and dopamine neurons are regulated, partly because of anatomical complexity. Here, Ogawa et al. used a modified rabies virus to systematically map direct inputs to serotonin neurons and found a surprising similarity of inputs between a group of serotonin neurons and a group of dopamine neurons. These results give a global picture of serotonin and dopamine neural circuits.
  • Global Scaling Down of Excitatory Postsynaptic Responses in Cerebellar Purkinje Cells Impairs Developmental Synapse Elimination

    • Shinya Kawata,
    • Taisuke Miyazaki,
    • Maya Yamazaki,
    • Takayasu Mikuni,
    • Miwako Yamasaki,
    • Kouichi Hashimoto,
    • Masahiko Watanabe,
    • Kenji Sakimura,
    • Masanobu Kano
    Kawata et al. examined how global scaling down of excitatory postsynaptic responses in mouse cerebellar Purkinje cells (PCs) affects developmental elimination of redundant climbing fiber (CF) synapses. Scaling down did not affect the early phase of CF elimination, but impaired the translocation of the strongest CF to PC dendrites and the late phase of CF elimination that requires Ca2+-dependent activation of Arc in PCs. The results indicate that proper synaptic scaling is crucial for accomplishing developmental synapse elimination.
  • Dysregulated Expression of Neuregulin-1 by Cortical Pyramidal Neurons Disrupts Synaptic Plasticity

    • Amit Agarwal,
    • Mingyue Zhang,
    • Irina Trembak-Duff,
    • Tilmann Unterbarnscheidt,
    • Konstantin Radyushkin,
    • Payam Dibaj,
    • Daniel Martins de Souza,
    • Susann Boretius,
    • Magdalena M. Brzózka,
    • Heinz Steffens,
    • Sebastian Berning,
    • Zenghui Teng,
    • Maike N. Gummert,
    • Martesa Tantra,
    • Peter C. Guest,
    • Katrin I. Willig,
    • Jens Frahm,
    • Stefan W. Hell,
    • Sabine Bahn,
    • Moritz J. Rossner,
    • Klaus-Armin Nave,
    • Hannelore Ehrenreich,
    • Weiqi Zhang,
    • Markus H. Schwab
    Altered levels of neuregulin-1 (NRG1) protein have been reported in the brain of schizophrenia patients. Agarwal et al. generated mouse mutants with reduced and elevated NRG1 levels and showed that both loss and gain of NRG1 function resulted in imbalanced excitatory-inhibitory neurotransmission, reduced synaptic plasticity, impaired sensorimotor gating, and protection against MK801-induced hyperactivity. Additionally, elevated NRG1 levels lead to enlarged ventricles and abnormal spine growth. The authors conclude that an “optimal” level of NRG1 signaling is essential for proper brain functions.
  • Modifying Lipid Rafts Promotes Regeneration and Functional Recovery

    • Nardos G. Tassew,
    • Andrea J. Mothe,
    • Alireza P. Shabanzadeh,
    • Paromita Banerjee,
    • Paulo D. Koeberle,
    • Rod Bremner,
    • Charles H. Tator,
    • Philippe P. Monnier
    Neuronal regeneration is very poor following CNS injuries. This is mostly due to the presence of extracellular proteins, such as RGMa, that hamper regeneration. In this study, Tassew et al. show that Neogenin, a receptor for RGMa, requires association with cholesterol-rich cell membrane domains to induce neuronal death and block axonal regeneration. They show that blocking Neogenin association with these membrane compartments allows for functional recovery following CNS injuries.
  • The TSC Complex Is Required for the Benefits of Dietary Protein Restriction on Stress Resistance In Vivo

    • Eylul Harputlugil,
    • Christopher Hine,
    • Dorathy Vargas,
    • Lauren Robertson,
    • Brendan D. Manning,
    • James R. Mitchell
    Harputlugil et al. now show a genetic requirement for the mTORC1 repressor TSC complex in improved insulin sensitivity and protection from acute surgical stress mediated by short-term dietary protein restriction (PR). One week of PR improved hepatic insulin sensitivity and preconditioned against hepatic ischemia reperfusion injury in wild-type mice but not liver-specific Tsc1 knockout mice. Mechanistically, increased prosurvival insulin signaling after reperfusion, facilitated by improved insulin sensitivity prior to ischemia, was partially required for PR-mediated protection from injury.
  • Reconstruction of Insulin Signal Flow from Phosphoproteome and Metabolome Data

    • Katsuyuki Yugi,
    • Hiroyuki Kubota,
    • Yu Toyoshima,
    • Rei Noguchi,
    • Kentaro Kawata,
    • Yasunori Komori,
    • Shinsuke Uda,
    • Katsuyuki Kunida,
    • Yoko Tomizawa,
    • Yosuke Funato,
    • Hiroaki Miki,
    • Masaki Matsumoto,
    • Keiichi I. Nakayama,
    • Kasumi Kashikura,
    • Keiko Endo,
    • Kazutaka Ikeda,
    • Tomoyoshi Soga,
    • Shinya Kuroda
    Cellular homeostasis is regulated by signals that are transmitted across multiple “omic” layers. However, molecular networks bridging these layers have not been identified comprehensively. Here, Yugi et al. describe comprehensive reconstruction of the molecular network between phosphoproteomic and metabolomic layers of rat hepatoma cells that is responsible for acute insulin action. Analysis of the network reveals the global landscape of insulin-signaling-dependent metabolic control and a novel mode of regulation of glycolysis by phosphorylation of liver-type phosphofructokinase-1.
  • Conversion of the Thymus into a Bipotent Lymphoid Organ by Replacement of Foxn1 with Its Paralog, Foxn4

    • Jeremy B. Swann,
    • Annelies Weyn,
    • Daisuke Nagakubo,
    • Conrad C. Bleul,
    • Atsushi Toyoda,
    • Christiane Happe,
    • Nikolai Netuschil,
    • Isabell Hess,
    • Annette Haas-Assenbaum,
    • Yoshihito Taniguchi,
    • Michael Schorpp,
    • Thomas Boehm
    The thymus is a lymphoid organ unique to vertebrates and required for T cell development. Its microenvironment is distinguished by the expression of the Foxn1 gene, a vertebrate-specific paralog of its metazoan ancestor, Foxn4. Swann et al. created a hypothetical primordial version of the thymus by replacing Foxn1 with Foxn4. Surprisingly, the reconstructed ancestral thymic microenvironment supported coincident but spatially segregated T and B cell development. These results point to the roots and chart the subsequent evolutionary trajectory of the thymus.
  • Multilineage Potential and Self-Renewal Define an Epithelial Progenitor Cell Population in the Adult Thymus

    • Kahlia Wong,
    • Natalie L. Lister,
    • Marco Barsanti,
    • Joanna M.C. Lim,
    • Maree V. Hammett,
    • Danika M. Khong,
    • Christopher Siatskas,
    • Daniel H.D. Gray,
    • Richard L. Boyd,
    • Ann P. Chidgey
    The thymus is responsible for T cell development and central tolerance but degenerates with age and is damaged by cytoablative treatments associated with cancer therapy. A central feature is the loss in thymic epithelial cells. Here, Wong et al. identify endogenous thymic epithelial stem/progenitor cells in the adult thymus within a major-histocompatibility-complex-II-low and α6-integrin/Sca-1-high subset. These findings will enable progress in understanding thymic epithelial cell aging and in developing strategies for endogenous thymus repair for immune regeneration.
  • Kcnn4 Is a Regulator of Macrophage Multinucleation in Bone Homeostasis and Inflammatory Disease

    • Heeseog Kang,
    • Audrey Kerloc’h,
    • Maxime Rotival,
    • Xiaoqing Xu,
    • Qing Zhang,
    • Zelpha D’Souza,
    • Michael Kim,
    • Jodi Carlson Scholz,
    • Jeong-Hun Ko,
    • Prashant K. Srivastava,
    • Jonathan R. Genzen,
    • Weiguo Cui,
    • Timothy J. Aitman,
    • Laurence Game,
    • James E. Melvin,
    • Adedayo Hanidu,
    • Janice Dimock,
    • Jie Zheng,
    • Donald Souza,
    • Aruna K. Behera,
    • Gerald Nabozny,
    • H. Terence Cook,
    • J.H. Duncan Bassett,
    • Graham R. Williams,
    • Jun Li,
    • Agnès Vignery,
    • Enrico Petretto,
    • Jacques Behmoaras
    Kang et al. establish the genetic determinants of macrophage multinucleation, which is involved in the formation of osteoclasts in bone or multinucleated giant cells during inflammatory reaction. They identify genetic variation on rat chromosome 9 linked to the expression of Kcnn4 as part of a macrophage multinucleation gene network. Kcnn4 is a calcium-activated potassium channel, and the authors show that it regulates macrophage multinucleation and bone mass. Blockade of Kcnn4 results in reduced susceptibility to glomerulonephritis and arthritis.


  • Sequencing of Captive Target Transcripts Identifies the Network of Regulated Genes and Functions of Primate-Specific miR-522

    • Shen Mynn Tan,
    • Rory Kirchner,
    • Jingmin Jin,
    • Oliver Hofmann,
    • Larry McReynolds,
    • Winston Hide,
    • Judy Lieberman
    Identifying miRNA-regulated genes is key to understanding miRNA function. Here, Tan et al. developed an unbiased method to identify with high specificity the target genes, miRNA recognition elements (MREs), and functions of primate-specific miR-522. Transcripts pulled down (without crosslinking) with a transfected biotinylated miRNA were sequenced to identify miRNA-regulated genes. Addition of RNase treatment identified MREs. Bioinformatics analysis of the transcription factors that regulate the genes, together with gene ontology analysis, provided a straightforward path toward uncovering miRNA function.