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 depolarizationmediated change of intracellular calcium level in the VMderived 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.

Foxa2 acts as a co-activator potentiating expression of the Nurr1-induced DA phenotype via epigenetic regulation

“…Foxa2 and/or a Nurr1-Foxa2 complex facilitate further opening of the chromatin-DNA structure, making it more accessible to other transcription factors and RNA polymerase. This gradual unzipping of histone-DNA contact may be a common mechanism of allowing first-bound transcription factors and co-activators to interact (Adams and Workman, 1995).Nurr1 interaction with an epigenetic repressor, CoREST, has been shown at inflammatory cytokine (Saijo et al, 2009) and DA phenotype promoters (He et al, 2011). We observed Nurr1 binding to CoREST on Th and Dat promoter regions in the absence of Foxa2.…”

“…Histone deacetylases (Hdacs) are epigenetic regulators of nuclear receptor-dependent differentiation (Nebbioso et al, 2010). In addition, Hdac1 is a common component of the CoREST-mediated epigenetic repressor complex (He et al, 2011;Saijo et al, 2009). Similar to CoREST, Hdac1 was also recruited to the Th and Dat promoter regions, and Hdac1 recruitment was significantly reduced in the presence of Foxa2 (Fig.…”

Generation of Dopamine Neurons from Rodent Fibroblasts through the Expandable Neural Precursor Cell Stage

“…The PCR conditions are provided in Table 1. Realtime PCR analyses were performed as described previously (14). Real-time PCR was performed on a CFX96 real-time system using iQ SYBR Green supermix (Bio-Rad).…”

“…Immunostaining of Cultured Cells and Brain TissuesImmunostaining of cultured cells and brain tissues was performed as described previously (14). Cells were photographed using epifluorescence and a confocal microscope (Leica Microsystems, Wetzlar, Germany).…”

“…In Vivo Transplantation and Histological Procedures-In vivo transplantation and histological procedures were performed as described previously (14). iNPCs were deposited at two sites (coordinates in anterior/posterior, medial/lateral, and dorsal/ventral relative to the bregma and dura: 1) ϩ0.07, Ϫ0.30, Ϫ0.55; 2) Ϫ0.1, Ϫ0.40, Ϫ0.50; incisor bar set at 3.5 mm below 0) while the animal was under anesthesia induced by Zoletil 50 (3.3 mg/kg; Virbac Laboratories, Carros, France) mixed with Rompun (93.3 g/kg; Bayer Korea, Seoul, Korea).…”