Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro

Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells

Epigenetic regulation via epigenetic factors in collaboration with tissue-specific transcription factors is curtail for establishing functional organ systems during development. Brain development is tightly regulated by epigenetic factors, which are coordinately activated or inactivated during processes, and their dysregulation is linked to brain abnormalities and intellectual disability. However, the precise mechanism of epigenetic regulation in brain development and neurogenesis remains largely unknown. Here, we show that Tip60/KAT5 deletion in neural stem/progenitor cells (NSCs) in mice results in multiple abnormalities of brain development. Tip60-deficient embryonic brain led to microcephaly, and proliferating cells in the developing brain were reduced by Tip60 deficiency. In addition, neural differentiation and neuronal migration were severely affected in Tip60-deficient brains. Following neurogenesis in developing brains, gliogenesis started from the earlier stage of development in Tip60-deficient brains, indicating that Tip60 is involved in switching from neurogenesis to gliogenesis during brain development. It was also confirmed in vitro that poor neurosphere formation, proliferation defects, neural differentiation defects, and accelerated astrocytic differentiation in mutant NSCs are derived from Tip60-deficient embryonic brains. This study uncovers the critical role of Tip60 in brain development and NSC maintenance and function in vivo and in vitro

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

<div><p>The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in <i>ZnT1</i>, <i>Metallothionein</i> (<i>MT</i>) and <i>ZnT4</i> genes (<i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.</p></div

Homeostasis of the secretory pathway is not significantly impaired in ZnT1^−/−MT^−/−ZnT4^−/− cells.

<p>(<b>A</b>) Surface IgM expression was not impaired in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells were fixed and immunostained without permeabilization. (<b>B</b>) The cell surface proteins biotinylated with membrane-impermeable reagent were almost the same between WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Surface refers to the solubilized proteins captured using streptavidin beads, while input refers to the aliquot of the biotinylated proteins before avidin capture (that is total cell lysate). In the surface panel, lectin blotting using WGA detects cell surface glycosylated proteins, and CBB staining detects total cell surface proteins. 1M4; <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. (<b>C</b>) Secretory <i>Cypridina</i> luciferase expression was not significantly different between WT and <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells. Both cells transiently transfected with secretory <i>Cypridina</i> luciferase reporter were cultured for 4 h after the medium change. Relative activity of <i>Cypridina</i> luciferase in the spent medium is shown (the luciferase activity/total cellular proteins in WT cells is defined as 1). Each value is the mean ± SD of three independent experiments (N.S., not significant).</p

Restoration of resistance of <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells by expression of the indicated genes against high zinc toxicity.

<p>The relative contribution of hZnT1, mMt-I or hZnT4 re-expression in <i>ZnT1</i>^−/−<i>MT</i>^−/−<i>ZnT4</i>^−/− cells against zinc toxicity was determined by counting the number of cells after 72 h exposure to the indicated concentrations of ZnSO₄. The relative contribution of exogenous expression of hZnT2 or mutant hZnT1 or hZnT4 is also indicated. Relative values presented are evaluations of the averages of three independent experiments. ++: growing to confluence; +: less growth compared with ++ (20–50% relative to ++); -: not growing.</p