RNA sequencing (RNA-SEQ) of Dnmt1 and Dnmt3a conditional knockout mice

Purpose: Epigenetic regulation contributes to pathogenesis of neurondegenerative disease. We found that dis-regulation of a 242-gene subnetwork related to DNA methylation regulated neuronal differentiation are common in AD and HD. There are two DNA methyltransferases, DNMT1 and DNMT3A, in the subnetwork. DNMT1 is one of top hub genes in the network and likely to play a key regulatory role in the subnetwork. To validate that DNMT1 is a key regulator for the subnetwork, and DNMT3A not as one, we constructed two brain-specific conditional knockout mice. Methods: Cortices were dissected from Dnmt1 conditional knockout (CKO), Dnmt3a CKO, and respective littermate control mice at 18 weeks. RNA was isolated from three biological replicates for each genotype using TRIzol (Invitrogen) extraction and isopropanol precipitation. RNA samples were resuspended in water and further purified with RNeasy columns with on-column DNase treatment (Qiagen). RNA purity was assessed by measuring the A260/A280 ratio using a NanoDrop and RNA quality checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument according to the manufacturer's instructions. Sequence reads were aligned to mouse genome assembly mm10 and quantified using Cufflinks. Results: Genes differentially expressed in CKO mice compared to littermate control mice, or the Dnmt1 CKO signature, including GSN (the genes with the largest number of connection in the subnetwork) and SOX10 (a key transcription factor of myelination), significantly overlaps with the subnetwork. Broadly, the Dnmt1 CKO signature is enriched for genes involved in GO biological processes immune response, lipid metabolism, endocytosis, glial cell differentiation, and nerve ensheatment, consistent with biological function of the subnetwork. Moreover, the Dnmt1 CKO mice show increased predilection to seizures, an incidence also increased in patients with AD (Amatniek et al, 2006) as well as juvenile form of HD (Cloud et al, 2012). In contrast, the Dnmt3a CKO signature does not overlap with the subnetwork (p=0.07) and is not enriched for any GO biological process. Conclusions: These results suggest the Dnmt1 regulates common genes related with AD and HD while Dnmt3a has little effect. All of the mice used in this study were handled in accordance with IACUC-approved protocols. Dnmt1flox/flox (Fan et al, 2001; Jackson-Grusby et al, 2001) and Dnmt3aflox/flox (Nguyen et al, 2007) mice were backcrossed onto a C57BL/6 background and crossed with Olig1-cre mice to generate Dnmt1 conditional knockout (Olig1cre/+;Dnmt1flox/flox) and littermate control (Olig1+/+;Dnmt1flox/flox) mice, and Dnmt3a conditional knockout (Olig1cre/+;Dnmt3aflox/flox) and littermate control (Olig1+/+;Dnmt3aflox/flox) mice.研究目的：表观遗传调控（epigenetic regulation）参与神经退行性疾病的发病机制。本研究发现，一个与DNA甲基化（DNA methylation）调控神经元分化相关的242基因子网（subnetwork）的失调，在阿尔茨海默病（Alzheimer's Disease, AD）和亨廷顿舞蹈病（Huntington's Disease, HD）中普遍存在。该子网包含两种DNA甲基转移酶（DNA methyltransferases）：DNMT1与DNMT3A。其中DNMT1是该网络的核心枢纽基因（hub genes）之一，极可能在该子网中发挥关键调控作用。为验证DNMT1是该子网的关键调控因子，而DNMT3A并非如此，我们构建了两种脑特异性条件性敲除（conditional knockout, CKO）小鼠。 实验方法：于18周龄时，分别从Dnmt1条件性敲除（CKO）、Dnmt3a条件性敲除（CKO）小鼠及其同窝对照小鼠中分离大脑皮层。采用TRIzol（Invitrogen）试剂提取并通过异丙醇沉淀法，从每种基因型的3个生物学重复样本中分离总RNA。将RNA样品重悬于无酶水中，随后使用带柱上DNase处理的RNeasy柱（Qiagen）进行进一步纯化。采用NanoDrop分光光度计检测A260/A280比值以评估RNA纯度，并使用Agilent 2100生物分析仪（Agilent Technologies）检测RNA完整性。按照TruSeq RNA样本制备试剂盒（Illumina）的操作规程，每份样品取约250 ng总RNA进行文库构建，并使用Illumina HiSeq 2500测序仪按照厂商提供的说明书完成测序。将测序读数比对至小鼠基因组组装版本mm10，并使用Cufflinks软件进行基因表达定量分析。 实验结果：与同窝对照小鼠相比，Dnmt1 CKO小鼠中差异表达的基因（即Dnmt1 CKO特征基因集），包括GSN（该子网中连接数最多的基因）和SOX10（髓鞘形成的关键转录因子），与该子网存在显著的基因重叠。整体而言，Dnmt1 CKO特征基因集显著富集于以下基因本体（Gene Ontology, GO）生物过程：免疫应答、脂质代谢、内吞作用、神经胶质细胞分化以及神经纤维髓鞘包被，与该子网的生物学功能高度一致。此外，Dnmt1 CKO小鼠表现出癫痫易感性升高，而AD患者（Amatniek et al, 2006）以及青少年型HD患者（Cloud et al, 2012）的癫痫发生率同样显著升高。与之相反，Dnmt3a CKO特征基因集与该子网无显著重叠（p=0.07），且未富集任何显著的GO生物过程。 研究结论：上述结果表明，Dnmt1调控与AD和HD相关的共有基因，而Dnmt3a几乎无此调控作用。本研究中所有实验动物的饲养与处理均符合经动物实验伦理委员会（Institutional Animal Care and Use Committee, IACUC）批准的实验方案。Dnmt1flox/flox（Fan et al, 2001; Jackson-Grusby et al, 2001）与Dnmt3aflox/flox（Nguyen et al, 2007）小鼠均经回交至C57BL/6遗传背景，并与Olig1-cre工具小鼠杂交，以获得Dnmt1条件性敲除（Olig1cre/+;Dnmt1flox/flox）及其同窝对照（Olig1+/+;Dnmt1flox/flox）小鼠，以及Dnmt3a条件性敲除（Olig1cre/+;Dnmt3aflox/flox）及其同窝对照（Olig1+/+;Dnmt3aflox/flox）小鼠。