Dynamic expression of MEIS1 homeoprotein in E14.5 forebrain and differentiated forebrain-derived neural stem cells

Barber, Benjamin A.; Liyanage, Vichithra R. B.; Zachariah, Robby M.; Olson, Carl O.; Bailey, Melissa A. G.; Rastegar, Mojgan

doi:10.1016/j.aanat.2013.04.005

Cited by 36 publications

(45 citation statements)

References 61 publications

(79 reference statements)

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“…RNA was converted to cDNA with a reaction set up containing Superscript III Reverse Transcriptase (Invitrogen) using previously described methods (Kobrossy et al, 2006;Liyanage et al, 2013;Nolte et al, 2006;Rastegar et al, 2004). Quantitative RT-PCR was performed using SYBR Green-based RT 2 qPCR Master Mix (Applied Biosystems) in a Fast 7500 Real-Time PCR machine (Applied Biosystems) as previously described (Barber et al, 2013;Liyanage et al, 2013;Olson et al, 2014). Transcript levels of Mecp2 and cell type-specific markers were examined using primers listed in Table 1.…”

Section: Quantitative Real Time Polymerase Chain Reaction (Qrt-pcr)mentioning

confidence: 99%

“…To assess the impact of ethanol exposure on Mecp2/MeCP2 transcript and protein expression, we used a previously characterized embryonic NSC differentiation system (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). Brain-derived neural stem cells were isolated from dissected forebrains of E14.5 mouse embryos and were cultured for 7 days in the presence of growth factors to generate neurospheres.…”

Section: A Differentiating Neural Stem Cell Model Was Used To Study Tmentioning

confidence: 99%

“…Western blotting was performed as previously described (Barber et al, 2013;Rastegar et al, 2000;Wu et al, 2001;. NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific) was used to extract nuclear proteins as previously described Olson et al, 2014).…”

Section: Western Blot (Wb)mentioning

confidence: 99%

“…Neural stem cells isolated from the forebrains of embryonic day (E) 14.5 CD-1 mice were cultured as previously described (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). In brief, dissected forebrain tissues were homogenized in NSC media Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 1:1 (DMEM/F12; Wisent) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glutamine, antibiotic/antimycotic, glucose, recombinant human epidermal growth factor (rhEGF; Sigma, 20 ng/ml), basic fibroblast growth factor (bFGF; Upstate, 20 ng/ml), heparin (Sigma, 2 μg/ml) and hormone mix [DMEM:F12, glucose (0.6%), insulin (0.25 mg/ml), transferrin (1 mg/ml), progesterone (0.2 μM), putrescine (0.097 mg/ml), sodium selenite (0.3 μM)] and plated at a density of 10 5 cells/cm 2 in serum-free full NSC media.…”

Section: Neural Stem Cell Isolation Culture and Differentiationmentioning

confidence: 99%

“…We have used this neural stem cell model to study the regulation of genes involved in neural development including Mecp2 and Meis1 (Barber et al, 2013;Liyanage et al, 2013;Rastegar et al, 2009). We have successfully used the same NSC system to rescue the aberrant neuronal morphologies in Mecp2-deficient NSC by MECP2 gene therapy strategies .…”

Section: Introductionmentioning

confidence: 99%

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Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

Liyanage

Zachariah

Davie

et al. 2015

Experimental Neurology

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Methyl CpG Binding Protein 2 (MeCP2) is an important epigenetic factor in the brain. MeCP2 expression is affected by different environmental insults including alcohol exposure. Accumulating evidence supports the role of aberrant MeCP2 expression in ethanol exposureinduced neurological symptoms. However, the underlying molecular mechanisms of ethanolinduced MeCP2 deregulation remain elusive. To study the effect of ethanol on Mecp2/MeCP2 expression during neurodifferentiation, we established an in vitro model of ethanol exposure, using differentiating embryonic brain-derived neural stem cells (NSC). Previously, we demonstrated the impact of DNA methylation at the Mecp2 regulatory elements (REs) on Mecp2/ MeCP2 expression in vitro and in vivo. Here, we studied whether altered DNA methylation at these REs is associated with the Mecp2/MeCP2 misexpression induced by ethanol. Binge-like and continuous ethanol exposure upregulated Mecp2/MeCP2, while ethanol withdrawal downregulated its expression. DNA methylation analysis by methylated DNA immunoprecipitation indicated that increased 5-hydroxymethylcytosine (5hmC) and decreased 5-methylcytosine (5mC) enrichment at specific REs were associated with upregulated Mecp2/MeCP2 following continuous ethanol exposure. The reduced Mecp2/MeCP2 expression upon ethanol withdrawal was associated with reduced 5hmC and increased 5mC enrichment at these REs. Moreover, ethanol altered global DNA methylation (5mC and 5hmC). Under the tested conditions, ethanol had minimal effects on Authors' contributionsVichithra R.B. Liyanage: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. Robby M. Zachariah: Conception and design, collection and assembly of data, and final approval of manuscript. James R. Davie: Scientific input, manuscript revisions, and final approval of manuscript. Mojgan Rastegar: Conception and design, contributed reagents/materials/analysis tools, data analysis and interpretation, manuscript writing, and final approval of manuscript. Conflict of interest statement

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