Zinc finger homeobox-3 (ZFHX3) exerts genome-wide regulation of dailygene expression in the suprachiasmatic nucleus [ChIP-seq]. Zinc finger homeobox-3 (ZFHX3) exerts genome-wide regulation of dailygene expression in the suprachiasmatic nucleus [ChIP-seq]

The mammalian suprachiasmatic nucleus (SCN), situated in the ventral hypothalamus, directs daily cellular and physiological 24-hr rhythms across the body. The cell-autonomous timing mechanism is based on a self-sustaining transcriptional-translational feedback loop (TTFL) that in turn co-ordinates the expression of clock-controlled genes (CCGs) that direct circadian programmes of SCN cellular activity. In the mouse, the transcription factor, ZFHX3 (zinc finger homeobox-3), is necessary for the development of the SCN and influences circadian behaviour in the adult. The molecular mechanisms by which ZFHX3 affects the SCN at transcriptome and genome levels are, however, poorly defined. Here, we used chromatin immunoprecipitation (ChIP-seq) to map the genomic localization of ZFHX3 sites in the SCN. In parallel, we conducted a comprehensive SCN RNA sequencing at six distinct times-of-day, and then compared the transcriptional profile between the control and ZFHX3-conditional null mutants. We show that the genome wide occupancy of ZFHX3 occurs predominantly around the gene transcription start site (TSS), co-localizing with the known histone modifications, and preferentially partnering with the core-clock TFs (CLOCK, BMAL1) to regulate the clock gene(s) transcription. Correspondingly, we show that the conditional loss of ZFHX3 in the adult has a dramatic effect on the SCN transcriptome, reducing the level of neuropeptides-encoding transcripts and abolishing rhythmic (24-h) oscillation of the clock TF, Bmal1. In addition, various TTFL genes and CCGs exhibited altered circadian expression profiles, consistent with the advanced daily behaviour under 12hr light- 12hr dark conditions. Taken together, these findings reveal the extensive genome-wide regulation conferred by ZFHX3 in the central clock that orchestrates daily timekeeping in mammals. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for ZFHX3 using the SCN tissues from WT C57BL/6J mice.

哺乳动物视交叉上核（suprachiasmatic nucleus, SCN）位于下丘脑腹侧，负责调控全身细胞与生理层面的每日24小时节律。细胞自主计时机制依托于自我维持的转录-翻译反馈环（transcriptional-translational feedback loop, TTFL），该反馈环可协调时钟调控基因（clock-controlled genes, CCGs）的表达，进而指导视交叉上核细胞活动的昼夜程序。在小鼠中，转录因子ZFHX3（锌指同源框3，zinc finger homeobox-3）对视交叉上核的发育必不可少，同时会影响成年个体的昼夜行为。然而，ZFHX3在转录组与基因组层面影响视交叉上核的分子机制目前仍尚不明确。
本研究通过染色质免疫沉淀测序（chromatin immunoprecipitation-sequencing, ChIP-seq）定位了视交叉上核内ZFHX3结合位点的基因组位置。同时，我们在六个不同的时间点对小鼠视交叉上核开展了全面的RNA测序，并对比了对照组与ZFHX3条件性敲除突变体的转录谱。
研究显示，ZFHX3的全基因组结合位点主要富集于基因转录起始位点（transcription start site, TSS）附近，与已知的组蛋白修饰共定位，并优先与核心时钟转录因子（CLOCK、BMAL1）协同调控时钟基因的转录。
相应地，本研究证实成年个体中ZFHX3的条件性缺失对视交叉上核的转录组产生了显著影响：降低了编码神经肽的转录本水平，并消除了时钟转录因子Bmal1的节律性（24小时）振荡。
此外，多种转录-翻译反馈环基因与时钟调控基因的昼夜表达谱发生改变，这与12小时光照-12小时黑暗环境下提前出现的日常行为表型相符。
综上，这些发现揭示了ZFHX3在哺乳动物中枢时钟中广泛的全基因组调控作用，该中枢时钟负责协调哺乳动物的每日计时活动。
整体实验设计：使用野生型（wild type, WT）C57BL/6J小鼠的视交叉上核组织，针对ZFHX3开展染色质免疫沉淀测序（ChIP-seq）。