Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.

Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. Here we show that developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation.Adult stem cells maintain populations of highly differentiated but short-lived cells throughout the life of the organism. To maintain the critical balance between stem cell and differentiating cell populations, stem cells have a potential to divide asymmetrically, producing one stem and one differentiating cell (1). The asymmetric outcome of stem cell divisions can be specified by regulated spindle orientation, such that the two daughter cells are placed in different microenvironments that either specify stem cell identity (stem cell niche) or allow differentiation (2,3).Drosophila male germline stem cells (GSCs) are maintained through attachment to somatic hub cells, which constitute the stem cell niche. Hub cells secrete the signaling ligand Upd, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the neighboring germ cells to specify stem cell identity (4,5). Drosophila male GSCs normally divide asymmetrically, producing one stem cell, which remains attached to the hub, and one gonialblast, which initiates differentiation. This stereotyped asymmetric outcome is controlled by the orientation of the mitotic spindle in GSCs: The spindle lies perpendicular to the hub so that one daughter cell inherits the attachment to the hub, whereas the other is displaced away (6).

The myelin sheath allows axons to rapidly conduct action potentials in the vertebrate nervous system. Incompletely understood axonal signals activate specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cAMP can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we report that Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20, and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.During peripheral nervous system (PNS) development, promyelinating Schwann cells associate with one segment of an axon and differentiate into myelinating Schwann cells that iteratively wrap their membrane around an axonal segment to form the myelin sheath (1). Axonal signals transiently activate the expression of the transcription factor Oct6 in Schwann cells that will form myelin, and cAMP can mimic axonal contact in vitro (2,3). Oct6 regulates Krox20 expression (4), and both transcription factors are required for Schwann cells to initiate myelination (5-7). Neuregulin signals and their ErbB receptors are involved in regulation of Oct6 and Krox20 (8), but the signaling pathways in Schwann cells that regulate myelination are not well understood.In a genetic screen for zebrafish mutants with abnormalities in myelinated axons, we previously identified two allelic mutations, st49 and st63, in which Myelin basic protein (Mbp) expression was not observed in peripheral nerves (9). Central nervous system (CNS) Mbp expression and PNS axonal marker expression were unaffected (9; Fig. S1). Except for an enlargement of the ear that was evident at 5 days post fertilization (dpf), st49 homozygous mutant larvae were morphologically indistinguishable from wild-type and heterozygous siblings (Fig. S2). High-resolution mapping experiments placed the st49 mutation in a region of Linkage Group 20 (LG20) that contains g-protein coupled receptor 126 (gpr126), which encodes a member of the adhesion G-protein coupled receptor (GPCR)

Author Contributions G.M. designed and performed the experiments, analyzed and interpreted the data, and wrote the manuscript. M.A.M. designed and supervised the research, analyzed and interpreted the data, and wrote the manuscript. C.V.C. co-designed and assisted with high spatiotemporal resolution microscopy experiments. E.J.H. and J.R.P. co-designed and assisted with zebrafish experiments. N.M. and T.P. co-designed and performed the BBB-on-a-chip experiments. C.C.D. assisted with some of the experiments. L.I.Z. and D.E.I. provided zebrafish and BBB-on-a-chip models, respectively.

In order to investigate whether covalent histone modifications may be involved in early embryonic reprogramming events, changes in global levels of a series of histone tail modifications were studied during oocyte maturation and pre-implantation mouse development using indirect immunofluorescence and scanning confocal microscopy. Results showed that histone modifications could be classified into two strikingly distinct categories. The first contains stable `epigenetic' marks such as histone H3 lysine 9 methylation [Me(Lys9)H3], histone H3 lysine 4 methylation [Me(Lys4)H3] and histone H4/H2A serine 1 phosphorylation [Ph(Ser1)H4/H2A]. The second group contains dynamic and reversible marks and includes hyperacetylated histone H4, histone H3 arginine 17 methylation [Me(Arg17)H3] and histone H4 arginine 3 methylation [Me(Arg3)H4]). Our results also showed that removal of these marks in eggs and early embryos occurs during metaphase suggesting that the enzymes responsible for the loss of these modifications are probably cytoplasmic in nature. Finally, we provide data demonstrating that treatment of cellular histones with peptidylarginine deiminase (PAD) results in loss of staining for the histone H4 arginine 3 methyl mark, suggesting that PADs can reverse histone arginine methyl modifications.

SUMMARYDuring peripheral nerve development, each segment of a myelinated axon is matched with a single Schwann cell. Tight regulation of Schwann cell movement, proliferation and differentiation is essential to ensure that these glial cells properly associate with axons. ErbB receptors are required for Schwann cell migration, but the operative ligand and its mechanism of action have remained unknown. We demonstrate that zebrafish Neuregulin 1 (Nrg1) type III, which signals through ErbB receptors, controls Schwann cell migration in addition to its previously known roles in proliferation and myelination. Chimera analyses indicate that ErbB receptors are required in all migrating Schwann cells, and that Nrg1 type III is required in neurons for migration. Surprisingly, expression of the ligand in a few axons is sufficient to induce migration along a chimeric nerve constituted largely of nrg1 type III mutant axons. These studies also reveal a mechanism that allows Schwann cells to fasciculate axons regardless of nrg1 type III expression. Time-lapse imaging of transgenic embryos demonstrated that misexpression of human NRG1 type III results in ectopic Schwann cell migration, allowing them to aberrantly enter the central nervous system. These results demonstrate that Nrg1 type III is an essential signal that controls Schwann cell migration to ensure that these glia are present in the correct numbers and positions in developing nerves.

In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.

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