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Neurogenic to Gliogenic Fate Transition Perturbed by Loss of HMGB2

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Neurogenic to Gliogenic Fate Transition Perturbed by Loss of HMGB2 fnmol-10-00153 May 19, 2017 Time: 16:22 # 1 ORIGINAL RESEARCH published: 23 May 2017 doi: 10.3389/fnmol.2017.00153 Neurogenic to Gliogenic Fate Transition Perturbed by Loss of HMGB2 Robert Bronstein1,2, Jackson Kyle3, Ariel B. Abraham3 and Stella E. Tsirka1,3* 1 Program in Neuroscience, Stony Brook University, Stony Brook, NY, United States, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States, 3 Molecular and Cellular Pharmacology Graduate Program, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, United States Mouse cortical development relies heavily on a delicate balance between neurogenesis and gliogenesis. The lateral ventricular zone produces different classes of excitatory pyramidal cells until just before birth, when the production of astroglia begins to prevail. Epigenetic control of this fate shift is of critical importance and chromatin regulatory elements driving neuronal or astroglial development play an vital role. Different classes of chromatin binding proteins orchestrate the transcriptional repression of neuronal- specific genes, while allowing for the activation of astrocyte-specific genes. Through proteomic analysis of embryonic neural progenitor cells (NPCs) our group had previously identified high mobility group B2 (HMGB2), a chromatin protein dynamically expressed throughout embryonic development. In the current study using cultures of perinatal NPCs from HMGB2C=C and HMGB2−=− mice we discovered that vital elements of the polycomb group (PcG) epigenetic complexes polycomb repressive complexes 1 Edited by: and 2 (PRC1/2) were downregulated during the differentiation process of HMGB2- Marina Guizzetti, Oregon Health & Science University, null NPCs. These epigenetic changes led to downstream changes in specific histone United States modification levels, specifically the trimethylation of H3K27, and a subsequent shift Reviewed by: in the perinatal neurogenesis to gliogenesis fate transition. Collectively these results David Peter Gavin, demonstrate that chromatin binding proteins, such as HMGB2, can have significant Jesse Brown VA Medical Center, United States effects on the epigenetic landscape of perinatal neural stem/progenitor cells. Amul J. Sakharkar, Savitribai Phule Pune University, India Keywords: HMGB2, histone modification, postnatal neurogenesis, epigenetics, chromatin Carole Gwizdek, Centre National de la Recherche Scientifique, France INTRODUCTION *Correspondence: Stella E. Tsirka Neural stem/progenitor cells (NSPCs) persist in discrete areas of the postnatal mouse brain [email protected] (Abraham et al., 2013a). In the ventricular zone (VZ) and sub-ventricular zone (SVZ) of the lateral ventricles, NSPCs proceed through sustained waves of neurogenesis (∼E10–E17.5) and Received: 05 January 2017 gliogenesis (∼E17.5–p10). These peaks of differentiation are not mutually exclusive: a small glial Accepted: 05 May 2017 component is produced in the embryonic period, and a small neuronal component is generated Published: 23 May 2017 in the early postnatal period. There is, however, a delicate and time precise balance that has to Citation: be maintained—from the proper neuronal lamination of the cerebral cortex to the functional Bronstein R, Kyle J, Abraham AB integration of astrocytes and myelinating oligodendrocytes into the parenchymal tissue (Abraham and Tsirka SE (2017) Neurogenic et al., 2013b). to Gliogenic Fate Transition Perturbed ∼ by Loss of HMGB2. Cortical neurogenesis ( E10–E17.5) proceeds through a highly stereotyped cascade, whereby Front. Mol. Neurosci. 10:153. radial glial fibers serve as a migratory scaffold for the inside out lamination of the cortical plate doi: 10.3389/fnmol.2017.00153 (Cao et al., 2014). These bipolar progenitors extend their arbors to make contact with the glia Frontiers in Molecular Neuroscience| www.frontiersin.org 1 May 2017| Volume 10| Article 153 fnmol-10-00153 May 19, 2017 Time: 16:22 # 2 Bronstein et al. HMGB2 Modulates Neurogenesis limitans dorsally, and the VZ ventrally (Abraham et al., assess the functional contribution of murine HMGB2 in the 2013a). Radial glial endfeet therefore have extensive access regulation of PcG proteins and consequent downstream effects to signals from the meninges, as well as the cerebrospinal during the perinatal transition from cortical neurogenesis to fluid in the embryonic lateral ventricles. As neurogenesis gliogenesis. begins to wane toward the end of the embryonic period, this pool of VZ progenitors undergoes a genetic fate switch— giving rise to three primary brain-derived glial subtypes: (1) MATERIALS AND METHODS astrocytes, (2) oligodendrocytes, (3) NG2C cells (Corley and Kroll, 2015). Postnatally, NSPCs retract their apical fibers from Animals and Genotyping the pial surface, and become restricted to VZ/SVZ, where All animal experiments performed have been approved by they are supported by a vast plexus of blood vessels, all the and are in agreement with the guidelines of the Stony Brook while maintaining their ventricular contacts (Decoville et al., University (SBU) Institutional Animal Care and Use Committee 2001). (IACUC). HMGB2 null breeding pairs on a C57BL/6 background Specific genetic programs serve to underpin cell fate were obtained from Dr. Lorenza Ronfani (Core Facility for transitions in the brain, such as the perinatal shift from Conditional Mutagenesis, San Raffaele Scientific Institute, Milan, cortical neurogenesis to gliogenesis. Polycomb group (PcG) and Italy). The HMGB2 deficient mouse line was created by trithorax group (TrxG) proteins are well known epigenetic conventional gene targeting methods (Kokovay et al., 2010). mediators and play key roles in chromatin dynamics during Due to the sterility of the HMGB2 homozygous null males this period (Déjardin et al., 2005). First characterized in the (Kokovay et al., 2010), two breeding strategies were employed Drosophila embryo, they are critical in patterning the body for the following experiments: heterozygous females were crossed plan, as they bind to polycomb responsive elements (PREs) with heterozygous males or homozygous females crossed with to mediate their modulatory effects (Egan et al., 2013). Many heterozygous males. In instances where Mendelian ratios did not C=C proteins are associated with and contribute to PcG-modulated yield adequate numbers of HMGB2 controls (e.g., the second transcriptional regulation, and they are subdivided into two breeding scheme), age-matched controls were substituted from −=− complexes in the mouse, polycomb repressive complex 1 (PRC1) non-transgenic C57BL/6 breeding. The HMGB2 animals are and polycomb repressive complex 2 (PRC2). The mammalian smaller in size, but do not initially appear grossly phenotypically PRC2 includes three core proteins critical for gene repression: different than wild-type animals. enhancer of zeste 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12) (Gabellini et al., Primary Adherent Monolayer Culture of 2002)—a complex primarily involved in catalyzing the mono-, NSPCs di-, and tri-methylation of histone H3K27 (Gruber et al., 2011; Pups ranging in age from postnatal day 0 to postnatal day 2 Gonzales-Roybal and Lim, 2013). Combinatorial activity of PcG were used for all cell culture experiments. Brains were extracted and TrxG in stem and progenitor cells regulates cell-type- and placed in sterile Hank’s buffered salt solution (HBSS) specific fate acquisition and differentiation reversibly through containing 2% glucose. From each hemisphere—olfactory bulbs, the co-modification of gene promoters with both the “active” hippocampi, midbrain, cerebellum, and meninges were dissected H3K4me3 and “repressive” H3K27me3 modifications (He et al., away and discarded. The remaining brain regions, VZ, SVZ, 2005). striatum, and minimal underlying cortex, were placed in 5 mL After a shotgun proteomic screen of proliferating and of HBSS supplemented with 2% glucose. The tissue was differentiating mouse embryonic NSPCs, we reported that triturated in 1 mL of complete Neurocult neural progenitor the chromatin architectural protein high mobility group B2 cells (NPC) Basal Proliferation Media with the addition of (HMGB2) was expressed with a high degree of temporal 20 ng/mL of recombinant human epidermal growth factor (rh variability—leading us to further probe its role in NSPC EGF), 10 ng/mL recombinant human basic fibroblast growth dynamics (Hirabayashi et al., 2009; Hirabayashi and Gotoh, factor (rh bFGF), 0.2% heparin, and 5% penicillin/streptomycin. 2010). Dorsal switch protein 1 (DSP1), the Drosophila homolog The tissue suspension was spun down at 1500 rpm at room of HMGB2, has been classified as an enhancer of trithorax temperature. The subsequent tissue pellet was resuspended and polycomb (a group of proteins modulating the activity of in 200 ml of complete Neurocult NPC Basal Proliferation PcG and TrxG) (Huang, 2014), and shown to form multimeric Media with the addition of 20 ng/mL of rh EGF, 10 ng/mL complexes—serving as a prominent recruiter of polycomb rh bFGF, 0.2% heparin, and 5% penicillin/streptomycin. The repressive factors to specific PREs during switches in cell tissue was further triturated with a pipette tip until a fate (Hwang et al., 2014). Knockdown of EED, a critical uniform cell suspension was achieved. The cell suspension component of PRC2, has been demonstrated to result in a was filtered through a 40 mm nylon mesh cell strainer and delayed neurogenic to gliogenic fate shift in E11.5 NSPCs plated on six-well
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