title: Prolonged membrane depolarization enhances midbrain dopamine neuron differentiation via epigenetic histone modifications.
abstract: Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea.
authors.forename: Xi-Biao
authors.initials: XB
authors.lastname: He
authors.forename: Sang-Hoon
authors.initials: SH
authors.lastname: Yi
authors.forename: Yong-Hee
authors.initials: YH
authors.lastname: Rhee
authors.forename: Hyemin
authors.initials: H
authors.lastname: Kim
authors.forename: Yong-Mahn
authors.initials: YM
authors.lastname: Han
authors.forename: Suk-Ho
authors.initials: SH
authors.lastname: Lee
authors.forename: Hyunsu
authors.initials: H
authors.lastname: Lee
authors.forename: Chang-Hwan
authors.initials: CH
authors.lastname: Park
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Lee
authors.forename: Eric
authors.initials: E
authors.lastname: Richardson
authors.forename: Byung-Woo
authors.initials: BW
authors.lastname: Kim
authors.forename: Sang-Hun
authors.initials: SH
authors.lastname: Lee
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D019175:DNA Methylation / Q000235:genetics; D059290:Dopaminergic Neurons / Q000166:cytology* / Q000378:metabolism*; D044127:Epigenesis, Genetic / Q000235:genetics; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008636:Mesencephalon / Q000166:cytology*; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D006657:Histones; C497394:Mecp2 protein, rat; D051783:Methyl-CpG-Binding Protein 2; C539884:Nr4a2 protein, rat; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D002118:Calcium