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Greenberg, Michael

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Greenberg

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Michael

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Greenberg, Michael
Author's Publications: search.filters.isAuthorOfPublication.99fff397-75af-4a5b-84fc-a6c477fe49a6

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    Bidirectional Perisomatic Inhibitory Plasticity of a Fos Neuronal Network
    (Springer Science and Business Media LLC, 2020-12-09) Yap, Ee-Lynn; Pettit, Noah L.; Davis, Christopher; Nagy, M. Aurel; Harmin, David; Golden, Emily; Dagliyan, Onur; Lin, Cindy; Rudolph, Stephanie; Sharma, Nikhil; Griffith, Eric C.; Harvey, Christopher D.; Greenberg, Michael
    Behavioral experiences activate the Fos transcription factor (TF) in sparse populations of neurons that are critical for encoding and recalling specific events1-3. However, there is limited understanding of the mechanisms by which experience drives circuit reorganization to establish a network of Fos-activated cells. It is also unknown if Fos is required in this process beyond serving as a marker of recent neural activity and, if so, which of its many gene targets underlie circuit reorganization. Here we demonstrate that when mice engage in spatial exploration of novel environments, perisomatic inhibition of Fos-expressing hippocampal CA1 pyramidal neurons by parvalbumin (PV)-interneurons (INs) is enhanced, while perisomatic inhibition by cholecystokinin (CCK)-INs is weakened. This bidirectional modulation of inhibition is abolished when the function of the Fos TF complex is disrupted. Single-cell RNA-sequencing, ribosome-associated mRNA profiling, and chromatin analyses, combined with electrophysiology, reveal that Fos activates the transcription of Scg2 (secretogranin II), a gene that encodes multiple distinct neuropeptides, to coordinate these changes in inhibition. As PV- and CCK-INs mediate distinct features of pyramidal cell activity4-6, the Scg2-dependent reorganization of inhibitory synaptic input might be predicted to affect network function in vivo. Consistent with this prediction, hippocampal gamma rhythms and pyramidal cell coupling to CA1 theta are significantly altered with loss of Scg2. These findings reveal an instructive role for Fos and Scg2 in establishing a network of Fos-activated neurons via the rewiring of local inhibition to form a selectively modulated state. The opposing plasticity mechanisms on distinct inhibitory pathways may support the consolidation of memories over time.
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    Kinesin superfamily protein Kif26b links Wnt5a-Ror signaling to the control of cell and tissue behaviors in vertebrates
    (eLife Sciences Publications, Ltd, 2017) Susman, Michael W; Karuna, Edith P; Kunz, Ryan; Gujral, Taranjit S; Cantú, Andrea V; Choi, Shannon S; Jong, Brigette Y; Okada, Kyoko; Scales, Michael K; Hum, Jennie; Hu, Linda; Kirschner, Marc; Nishinakamura, Ryuichi; Yamada, Soichiro; Laird, Diana J; Jao, Li-En; Gygi, Steven; Greenberg, Michael; Ho, Hsin-Yi Henry
    Wnt5a-Ror signaling constitutes a developmental pathway crucial for embryonic tissue morphogenesis, reproduction and adult tissue regeneration, yet the molecular mechanisms by which the Wnt5a-Ror pathway mediates these processes are largely unknown. Using a proteomic screen, we identify the kinesin superfamily protein Kif26b as a downstream target of the Wnt5a-Ror pathway. Wnt5a-Ror, through a process independent of the canonical Wnt/β-catenin-dependent pathway, regulates the cellular stability of Kif26b by inducing its degradation via the ubiquitin-proteasome system. Through this mechanism, Kif26b modulates the migratory behavior of cultured mesenchymal cells in a Wnt5a-dependent manner. Genetic perturbation of Kif26b function in vivo caused embryonic axis malformations and depletion of primordial germ cells in the developing gonad, two phenotypes characteristic of disrupted Wnt5a-Ror signaling. These findings indicate that Kif26b links Wnt5a-Ror signaling to the control of morphogenetic cell and tissue behaviors in vertebrates and reveal a new role for regulated proteolysis in noncanonical Wnt5a-Ror signal transduction.
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    Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex
    (2017) Hrvatin, Sinisa; Hochbaum, Daniel; Nagy, M. Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon; Sabatini, Bernardo; Greenberg, Michael
    Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders.
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    Mapping the genomic landscape of inherited retinal disease genes prioritizes genes prone to coding and noncoding copy-number variations
    (Nature Publishing Group, 2018) Van Schil, Kristof; Naessens, Sarah; Van de Sompele, Stijn; Carron, Marjolein; Aslanidis, Alexander; Van Cauwenbergh, Caroline; Kathrin Mayer, Anja; Van Heetvelde, Mattias; Bauwens, Miriam; Verdin, Hannah; Coppieters, Frauke; Greenberg, Michael; Yang, Marty; Karlstetter, Marcus; Langmann, Thomas; De Preter, Katleen; Kohl, Susanne; Cherry, Timothy J; Leroy, Bart P; Lupski, James R; Carvalho, Claudia; van Min, Max; Klous, Petra; De Jaegere, Sarah; Hooghe, Sally; De Baere, Elfride
    Purpose Part of the hidden genetic variation in heterogeneous genetic conditions such as inherited retinal diseases (IRDs) can be explained by copy-number variations (CNVs). Here, we explored the genomic landscape of IRD genes listed in RetNet to identify and prioritize those genes susceptible to CNV formation. Methods: RetNet genes underwent an assessment of genomic features and of CNV occurrence in the Database of Genomic Variants and literature. CNVs identified in an IRD cohort were characterized using targeted locus amplification (TLA) on extracted genomic DNA. Results: Exhaustive literature mining revealed 1,345 reported CNVs in 81 different IRD genes. Correlation analysis between rankings of genomic features and CNV occurrence demonstrated the strongest correlation between gene size and CNV occurrence of IRD genes. Moreover, we identified and delineated 30 new CNVs in IRD cases, 13 of which are novel and three of which affect noncoding, putative cis-regulatory regions. Finally, the breakpoints of six complex CNVs were determined using TLA in a hypothesis-neutral manner. Conclusion: We propose a ranking of CNV-prone IRD genes and demonstrate the efficacy of TLA for the characterization of CNVs on extracted DNA. Finally, this IRD-oriented CNV study can serve as a paradigm for other genetically heterogeneous Mendelian diseases with hidden genetic variation.
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    Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement
    (National Academy of Sciences, 2018) Kalish, Brian; Cheadle, Lucas; Hrvatin, Sinisa; Nagy, M. Aurel; Rivera, Samuel; Crow, Megan; Gillis, Jesse; Kirchner, Rory; Greenberg, Michael
    Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.
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    A Chemical Genetic Approach Reveals Distinct Mechanisms of EphB Signaling During Brain Development
    (2012) Soskis, Michael J.; Ho, Hsin-Yi Henry; Bloodgood, Brenda L.; Robichaux, Michael A.; Malik, Athar; Ataman, Bulent; Rubin, Alex A.; Zieg, Janine; Zhang, Chao; Shokat, Kevan M.; Sharma, Nikhil; Cowan, Christopher W.; Greenberg, Michael
    EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, as EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events. To address these issues, we engineered triple knockin mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly, and specifically blocked. Using these mice we demonstrate that the tyrosine kinase activity of EphBs is required for axon guidance in vivo. By contrast, EphB-mediated synaptogenesis occurs normally when the kinase activity of EphBs is inhibited suggesting that EphBs mediate synapse development by an EphB tyrosine kinase-independent mechanism. Taken together, these experiments reveal that EphBs control axon guidance and synaptogenesis by distinct mechanisms, and provide a new mouse model for dissecting EphB function in development and disease.
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    Disruption of DNA methylation-dependent long gene repression in Rett syndrome
    (2015) Gabel, Harrison W.; Kinde, Benyam Z.; Stroud, Hume; Gilbert, Caitlin S.; Harmin, David; Kastan, Nathaniel R.; Hemberg, Martin; Ebert, Daniel H.; Greenberg, Michael
    Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.
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    Widespread transcription at neuronal activity-regulated enhancers
    (Nature Publishing Group, 2010) Kim, Tae-Kyung; Hemberg, Martin; Gray, Jesse; Costa, Allen M.; Bear, Daniel Marcus; Wu, Jing; Harmin, David; Laptewicz, Mike; Barbara-Haley, Kellie; Kuersten, Scott; Markenscoff-Papadimitriou, Eirene; Kuhl, Dietmar; Bito, Haruhiko; Worley, Paul F.; Kreiman, Gabriel; Greenberg, Michael
    We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified ,12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.
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    Unidirectional Eph/ephrin signaling creates a cortical actomyosin differential to drive cell segregation
    (The Rockefeller University Press, 2016) O’Neill, Audrey K.; Kindberg, Abigail A.; Niethamer, Terren K.; Larson, Andrew R.; Ho, Hsin-Yi Henry; Greenberg, Michael; Bush, Jeffrey O.
    Cell segregation is the process by which cells self-organize to establish developmental boundaries, an essential step in tissue formation. Cell segregation is a common outcome of Eph/ephrin signaling, but the mechanisms remain unclear. In craniofrontonasal syndrome, X-linked mosaicism for ephrin-B1 expression has been hypothesized to lead to aberrant Eph/ephrin-mediated cell segregation. Here, we use mouse genetics to exploit mosaicism to study cell segregation in the mammalian embryo and integrate live-cell imaging to examine the underlying cellular and molecular mechanisms. Our data demonstrate that dramatic ephrin-B1–mediated cell segregation occurs in the early neuroepithelium. In contrast to the paradigm that repulsive bidirectional signaling drives cell segregation, unidirectional EphB kinase signaling leads to cell sorting by the Rho kinase–dependent generation of a cortical actin differential between ephrin-B1– and EphB-expressing cells. These results define mechanisms of Eph/ephrin-mediated cell segregation, implicating unidirectional regulation of cortical actomyosin contractility as a key effector of this fundamental process.
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    Integrated Genome Analysis Suggests that Most Conserved Non-Coding Sequences are Regulatory Factor Binding Sites
    (Oxford University Press, 2012) Hemberg, Martin; Gray, Jesse; Cloonan, Nicole; Kuersten, Scott; Grimmond, Sean; Greenberg, Michael; Kreiman, Gabriel
    More than 98% of a typical vertebrate genome does not code for proteins. Although non-coding regions are sprinkled with short (<200 bp) islands of evolutionarily conserved sequences, the function of most of these unannotated conserved islands remains unknown. One possibility is that unannotated conserved islands could encode non-coding RNAs (ncRNAs); alternatively, unannotated conserved islands could serve as promoter-distal regulatory factor binding sites (RFBSs) like enhancers. Here we assess these possibilities by comparing unannotated conserved islands in the human and mouse genomes to transcribed regions and to RFBSs, relying on a detailed case study of one human and one mouse cell type. We define transcribed regions by applying a novel transcript-calling algorithm to RNA-Seq data obtained from total cellular RNA, and we define RFBSs using ChIP-Seq and DNAse-hypersensitivity assays. We find that unannotated conserved islands are four times more likely to coincide with RFBSs than with unannotated ncRNAs. Thousands of conserved RFBSs can be categorized as insulators based on the presence of CTCF or as enhancers based on the presence of p300/CBP and H3K4me1. While many unannotated conserved RFBSs are transcriptionally active to some extent, the transcripts produced tend to be unspliced, non-polyadenylated and expressed at levels 10 to 100-fold lower than annotated coding or ncRNAs. Extending these findings across multiple cell types and tissues, we propose that most conserved non-coding genomic DNA in vertebrate genomes corresponds to promoter-distal regulatory elements.