Abstract:
Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.
摘要:
人类细胞系模型,包括神经元前体系LUHMES,对于研究印迹区域内的发育转录动力学很重要,特别是15q11-q13Angelman(AS)和Prader-Willi(PWS)综合征基因座。AS是由于神经元中母体UBE3A丢失而导致的,其中父系等位基因被会聚的反义转录物UBE3A-ATS沉默,在非神经元中终止于PWAR1的lncRNA。qRT-PCR分析证实了UBE3A-ATS在分化LUHMES神经元中的排他性和进行性增加,验证其用于研究UBE3A沉默的用途。全基因组转录组分析揭示了神经元分化过程中11834个基因的变化,包括15q11-q13基因座内大多数基因的上调。为了确定与转录活性相关的染色质环的动态变化,我们执行了4C验证的HiChIP,它鉴定了MAGEL2-SNRPN和PWAR1-UBE3A之间的两个神经元特异性CTCF环。为了确定等位基因特异性差异甲基化区域(DMR)是否可能与CTCF环锚相关,进行了全基因组长读数纳米孔测序.我们确定了SNRPN上游环锚附近的父系低甲基化DMR,神经元和PWAR1CTCF锚附近的父系高甲基化DMR,未分化细胞。与神经元转录的增加一致。此外,在两种细胞类型中都观察到CTCF环锚附近的DMRs,指示调节印迹转录的染色质环的等位基因特异性差异。这些结果提供了LUHMES神经元分化过程中15q11-q13表观遗传景观的综合视图,强调转录的复杂相互作用,染色质循环,和DNA甲基化。他们还为AS和PWS的未来治疗方法提供了见解。