Epigenetic and transcriptional landscapes of Dnmt3a-deficient olfactory sensory neurons

During differentiation, neurons experience a reorganization of DNA modification patterns within their genomes. However, the mechanisms underlying this developmental patterning and its role in defining the neurona­­l state are currently unclear. Here, we find that the d­­e novo DNA methyltransferase Dnmt3a is necessary for elevated levels of 5-hydroxymethylcytosine (5hmC), a derivative of 5-methylcytosine (5mC), in olfactory sensory neurons (OSNs). Through an analysis of genome-wide 5mC and 5hmC distributions in isolated OSNs, we find that Dnmt3a-dependent 5mC and 5hmC occurs within regions of high accessibility, neural enhancers, and the transcription start sites of transcribed genes. Its loss results in the global disruption of gene expression patterns, including the upregulation of silent genes, the downregulation of mOSN-expressed genes, and the alteration of odorant-induced transcriptional responses of immediate early genes. Together, these results demonstrate that Dnmt3a is necessary to define the neuronal transcriptional state and may be broadly involved in refining expression profiles within differentiated cells. Overall design: To determine the contributions of Dnmt3a to the DNA modification and transcriptional landscapes of a post-mitotic neuronal population, we performed DNA immunoprecipitation (DIP-seq) using antibodies specific for 5mC and 5hmC and rRNA-depleted transcriptional profiling (RNA-seq) coupled to high-throughput sequencing using genomic DNA or RNA from FACS-isolated mature olfactory sensory neurons (mOSNs) from main olfactory epithelium (MOE) of Dnmt3a wildtype (WT), heterozygous-null (Het), or homozygous-null (KO) 3-week old mice. Similarly, to compare this information with other epigenetic features of the MOE, we performed H3K4me1 (WT), H3K27ac (WT), and H3K27me3 (WT and KO) chromatin immunoprecipitation (ChIP)-seq and DNase I hypersensitivity assays (DNase-seq) using MOE nuclei from 3-week old mice. In addition, we assayed the influence of Dnmt3a-deficiency on the induction of odorant-responsive genes by exposing 3-week old Dnmt3a WT, Het, and KO mice to either water or a 1:1:1 mixture of amyl acetate:acetophenone:octanal for 1 hour and performed rRNA-depleted RNA-seq using RNA isolated from their MOEs.

在神经元分化过程中，其基因组内的DNA修饰模式会发生重组。然而，这种发育模式背后的调控机制，以及其在定义神经元状态中的作用，目前仍不明确。本研究发现，从头DNA甲基转移酶 (de novo DNA methyltransferase) Dnmt3a是嗅觉感觉神经元 (olfactory sensory neurons, OSNs) 中5-羟甲基胞嘧啶 (5-hydroxymethylcytosine, 5hmC，其为5-甲基胞嘧啶 (5-methylcytosine, 5mC) 的衍生物) 水平升高所必需的。通过对分离得到的嗅觉感觉神经元中全基因组范围的5mC与5hmC分布进行分析，我们发现依赖于Dnmt3a的5mC和5hmC富集于高可及性区域、神经增强子以及转录基因的转录起始位点。Dnmt3a的缺失会导致基因表达模式的全局紊乱，包括沉默基因的上调、成熟嗅觉感觉神经元特异性表达基因的下调，以及即刻早期基因的气味诱导转录应答发生改变。综上，上述结果表明，Dnmt3a是定义神经元转录状态所必需的，且可能广泛参与细化分化细胞内的基因表达谱。

实验设计概述：为探明Dnmt3a对有丝分裂后神经元群体的DNA修饰与转录景观的调控作用，我们利用针对5mC和5hmC的特异性抗体开展了DNA免疫沉淀测序 (DNA immunoprecipitation-sequencing, DIP-seq) 实验，并针对从3周龄Dnmt3a野生型 (wildtype, WT)、杂合敲除 (heterozygous-null, Het) 或纯合敲除 (homozygous-null, KO) 小鼠的主嗅觉上皮 (main olfactory epithelium, MOE) 中通过荧光激活细胞分选 (fluorescence-activated cell sorting, FACS) 分离得到的成熟嗅觉感觉神经元 (mOSN) 的基因组DNA或RNA，开展了去核糖体RNA转录组测序 (RNA-seq)。

为将上述结果与主嗅觉上皮的其他表观遗传特征进行对比，我们利用3周龄小鼠的主嗅觉上皮细胞核开展了H3K4me1（野生型）、H3K27ac（野生型）、H3K27me3（野生型与纯合敲除型）染色质免疫沉淀测序 (chromatin immunoprecipitation-sequencing, ChIP-seq) 以及DNase I超敏性检测 (DNase-seq)。

此外，为探究Dnmt3a缺失对气味应答基因诱导的影响，我们将3周龄的Dnmt3a野生型、杂合敲除型及纯合敲除型小鼠分别暴露于纯水或体积比为1:1:1的乙酸戊酯、苯乙酮与辛醛混合气体中1小时，随后提取其主嗅觉上皮的RNA并开展去核糖体RNA转录组测序。