NPAS4 regulates the transcriptional response of suprachiasmatic nucleus neurons to photic input

In mammals, the hypothalamic suprachiasmatic nucleus (SCN) is the master circadian pacemaker. It is primarily entrained by environmental photic stimuli. However, the molecular bases of the response of heterogeneous cells in the SCN to photic stimuli is not fully understood. In this study, we combined bulk RNA/ChIP/single nuclei sequencing with circadian behavioral assays to investigate cellular identities in the SCN and explore how they react to photic stimuli. We identified 3 major peptidergic cells in the SCN, Avp-expressing, Vip-expressing and Cck-expressing cells. In each peptidergic cell type, there is a light responsive cell cluster, and each of these is enriched for Neuronal PAS Domain Protein 4 (NPAS4) target genes. In addition, Npas4 null mice have a 1hr longer circadian period in constant darkness and a damped phase responsive curve to photic stimuli. Bulk RNA seq in Npas4-/- SCN revealed a reduced magnitude of light induced gene expression. Together, our data indicate that NPAS4 is part of the machinery that orchestrates the molecular response to photic stimuli in the SCN. 1) Bulk RNA seq for light stimulation in wildtype mice: 2-4 replicates are involved in each condition. There are two dark control conditions, one is from CT17, the beginning of the light pulse and the other is from CT23, the end of the light pulse. In addition, there are 4 light pulse conditions, 0.5, 1, 3, and 6 hrs in length of light simulation. Each replicate contains 6 pairs of SCNs. 2) Bulk RNA seq for light stimulation in Npas4 null mice and control wild type littermates: 3 replicates are involved in each condition. There are 4 conditions total: wildtype with and without 1 hr light pulse at CT17 and Npas4-/- mice with and without 1 hr light pulse at CT17. Each replicate contains 6 pairs of SCNs. 3) ChIPseq: Two samples, each containing 2 pairs of SCN collected with or without 1 hr light stimulation. 4) Single nuclei sequencing: In total, 10 samples were analyzed. The first batch contains one dark control and one 1 hr light stimulation condition and sequencing was done by Illumina Hiseq. The second batch contains four dark control replicates and four 1 hr light stimulation replicates and sequencing was performed twice by Illumina NovaSeq.在哺乳动物体内，下丘脑视交叉上核（hypothalamic suprachiasmatic nucleus, SCN）是中枢昼夜节律起搏位点，其节律活动主要通过环境光刺激实现同步。然而，目前学界尚未完全阐明SCN内异质性细胞对光刺激产生应答的分子机制。本研究整合了批量RNA测序、染色质免疫沉淀（ChIP）测序与单细胞核测序技术，并结合昼夜节律行为学实验，旨在解析SCN内的细胞类群特征，并探究其对光刺激的应答机制。本研究在SCN内鉴定出3种主要的肽能细胞类群，分别为表达精氨酸加压素（Avp）、血管活性肠肽（Vip）以及胆囊收缩素（Cck）的细胞。在每一种肽能细胞类群中，均存在一个光应答细胞亚群，且该亚群均富集神经元PAS结构域蛋白4（Neuronal PAS Domain Protein 4, NPAS4）的靶基因。此外，Npas4基因敲除小鼠在持续黑暗环境下的昼夜节律周期延长1小时，且其对光刺激的相位应答曲线出现衰减。对Npas4-/-小鼠SCN进行的批量RNA测序结果显示，光诱导的基因表达幅度显著降低。综合以上结果，本研究数据表明NPAS4是调控SCN内光刺激分子应答过程的核心机制之一。 1) 野生型小鼠光刺激下的批量RNA测序：每组实验设置2~4次生物学重复。设置2组黑暗对照样本，分别采集于光照脉冲起始时点CT17以及光照脉冲结束时点CT23。此外设置4组光照脉冲实验条件，光照时长分别为0.5、1、3及6小时。每份样本包含6对SCN组织。 2) Npas4基因敲除小鼠及同窝野生型对照小鼠光刺激下的批量RNA测序：每组实验设置3次生物学重复。共设置4组实验条件：在CT17时点施加1小时光照脉冲的野生型小鼠、未施加光照脉冲的野生型小鼠、在CT17时点施加1小时光照脉冲的Npas4-/-小鼠，以及未施加光照脉冲的Npas4-/-小鼠。每份样本包含6对SCN组织。 3) ChIP测序：共设置2组样本，分别采集自施加1小时光照刺激与未施加光照刺激的小鼠，每组样本包含2对SCN组织。 4) 单细胞核测序：共分析10组样本。第一批实验包含1组黑暗对照与1组1小时光照刺激样本，测序平台为Illumina Hiseq。第二批实验包含4次黑暗对照生物学重复与4次1小时光照刺激生物学重复，测序平台为Illumina NovaSeq，且进行了两次重复测序。