A genome-wide search for epigenetically regulated genes in zebra finch using MethylCap-seq and RNA-seq. Taeniopygia guttata

DNA methylation is tightly linked with gene expression regulation and has long been regarded a stable epigenetic mark in postmitotic cells. However, it recently became clear that postnatal brains appear to show stimulus-induced de novo CpG methylation or active demethylation related to neuronal plasticity. Due to striking homologies between the brains of birds and mammals, songbirds, especially the zebra finch, propose an attractive model for investigating the genome-wide DNA methylation profile and DNA methylation reconfiguration during brain development. In order to obtain a first genome-wide compendium of genes under putative DNA methylation control, we performed MethyCap-seq experiments on two recently cultured zebra finch cell lines, G266 and ZFTMA, also upon AZA-induced demethylation. First, the MethylCap-seq methodology in zebra finch was validated by comparison with RRBS generated data. Subsequently, quantitative analysis identified 30,700 significantly demethylated loci upon AZA-treatment. Further examination revealed enrichment of these regions in exons and promoters. To assess the influence of methylation on gene expression, RNA-seq experiments were performed. Comparison of the RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed obvious enrichment for neurological networks. A subset of genes was validated using qPCR and CpG pyrosequencing. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control. Overall design: MethylCap-seq and RNA-seq experiments were performed on DMSO- and AZA-treated zebra finch cell lines, i.e. G266 and ZFTMA. As a quality control, also an untreated ZFTMA sample was analyzed with MethylCap-seq and RRBS.

DNA甲基化（DNA methylation）与基因表达调控紧密关联，长期以来被视作有丝分裂后细胞中稳定的表观遗传标记。然而近期研究表明，出生后的大脑中存在由刺激诱导的从头CpG甲基化或与神经可塑性相关的主动去甲基化过程。鉴于鸟类与哺乳动物大脑间存在显著同源性，鸣禽尤其是斑胸草雀，成为探究脑发育过程中全基因组DNA甲基化谱式与甲基化重编程的极具吸引力的模型系统。

为首次获取受推定DNA甲基化调控的基因的全基因组汇编数据集，我们对两株近期培养的斑胸草雀细胞系G266与ZFTMA开展了MethyCap-seq实验，同时设置了AZA诱导去甲基化的处理组。首先，通过与RRBS生成的数据进行比对，验证了斑胸草雀样本中MethyCap-seq方法的有效性。后续定量分析共鉴定出30700个经AZA处理后显著去甲基化的位点。进一步分析显示，这些去甲基化区域在外显子与启动子区域存在显著富集。

为评估甲基化对基因表达的影响，我们开展了RNA-seq实验。对比RNA-seq与MethyCap-seq的分析结果发现，在3457个经AZA上调的基因中，至少357个基因的转录受启动子区域甲基化的推定调控；通路分析显示该类基因显著富集于神经系统相关通路。我们采用qPCR与CpG焦磷酸测序对部分候选基因进行了实验验证。

据我们所知，本研究首次构建了斑胸草雀基因组的全基因组DNA甲基化图谱，并提供了一套完整的受推定甲基化调控转录的基因集合。

整体实验设计：分别对经DMSO与AZA处理的斑胸草雀细胞系G266、ZFTMA开展MethyCap-seq与RNA-seq实验。作为质量控制环节，我们还采用MethyCap-seq与RRBS对未处理的ZFTMA样本进行了检测分析。