دانلود رایگان مقاله انگلیسی خطر ژنتیکی بیماری پارکینسون در یک خط سلولی عصبی میان مغزی - الزویر 2018

ABSTRACT

In genome-wide association studies of complex diseases, many risk polymorphisms are found to lie in non-coding DNA and likely confer risk through allele-dependent differences in gene regulatory elements. However, because distal regulatory elements can alter gene expression at various distances on linear DNA, the identity of relevant genes is unknown for most risk loci. In Parkinson's disease, at least some genetic risk is likely intrinsic to a neuronal subpopulation of cells in the brain regions affected. In order to compare neuron-relevant methods of pairing risk polymorphisms to target genes as well as to further characterize a single-cell model of a neurodegenerative disease, we used the portionally-dopaminergic, neuronal, mesencephalic-derived cell line LUHMES to dissect differentiation-specific mechanisms of gene expression. We compared genome-wide gene expression in undifferentiated and differentiated cells with genome-wide histone H3K27ac and CTCF-bound regions. Whereas promoters and CTCF binding were largely consistent between differentiated and undifferentiated cells, enhancers were mostly unique. We matched the differentiation-specific appearance or disappearance of enhancers with changes in gene expression and identified 22,057 enhancers paired with 6388 differentially expressed genes by proximity. These enhancers are enriched with at least 13 transcription factor response elements, driving a cluster of genes involved in neurogenesis. We show that differentiated LUHMES cells, but not undifferentiated cells, show enrichment for PD-risk SNPs. Candidate genes for these loci are largely unrelated, though a subset is linked to synaptic vesicle cycling and transport, implying that PD-related disruption of these pathways is intrinsic to dopaminergic neurons.

4. Materials and methods

4.1. LUHMES culture

LUHMES cells were cultured essentially as done by (Scholz et al., 2011). Briefly, the cells were incubated in a humidified 37 °C, 5% CO2 incubator on flasks pre-coated with 50 mg/mL poly-L-ornithine (Sigma, Cat # P3655) and 1 mg/mL fibronectin (Sigma, Cat # F114) in water. The coated flasks were incubated at 37 °C overnight, rinsed with water, and allowed to dry before seeding cells. Cells were cultured in complete growth medium containing Advanced DMEM:F12 (Thermo Fisher, Cat # 12634-010) with 2 mM L-glutamine (Thermo Fisher, Cat # 25030081), 1X N-2 supplement (Thermo Fisher, Cat # 17502-048), and 0.04 mg/mL bFGF (Stemgent, Cat # 03-0002). Cells were allowed to reach 80% confluency before passaging with 0.025% trypsin/EDTA. Prior to differentiation, cells were seeded at 3.5 × 106 per T75 flask containing complete growth medium and incubated at 37 °C for 24 h. For induction of differentiation, culture medium was changed to freshly prepare DMEM:F12 with 2 mM L-glutamine, 1X N-2, 1 mM cAMP (Carbosynth, Cat # ND07996), 1 mg/mL tetracycline (Sigma, Cat # T7660), and 2 ng/mL glial cell line-derived neurotrophic factor (GDNF) (Sigma, Cat # G1777).