Class 3 PI3K participates in nuclear gene transcription and co-activates the circadian clock to promote de novo purine synthesis [RNA-seq]

Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient sensing signaling pathways, yet mechanisms of their interaction remain not fully understood. Here, surprisingly, we find that class 3 Phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in participating to gene transcription as a co-activator of heterodimeric transcription factor and circadian driver BMAL1-CLOCK. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between lipid kinase Vps34 and regulatory subunit Vps15. We find that although both subunits of class 3 PI3K interact with RNA Pol2 and co-localize with active transcription sites, exclusive loss of Vps15 blunted BMAL1-CLOCK transcriptional activity. Thus, we established non-redundancy between nuclear Vps34 and Vps15 reflected in a persistent nuclear pool of Vps15 in Vps34-depleted cells and ability of Vps15 to co-activate BMAL1-CLOCK independently of its complex with Vps34. In physiology, we find Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promoted pro-anabolic de novo purine nucleotide synthesis. We show Vps15 activated transcription of Ppat, a key enzyme for production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, BMAL1 recruitment to chromatin is unmodified but it coincides with depletion of Vps15 on the promoters of its targets, Nr1d1 and Ppat. Our findings suggest that nuclear pool of class 3 PI3K Vps15 subunit could couple transcriptional activity of the circadian clock for temporal regulation of energy homeostasis and it opens novel avenues for establishing the complexity for nuclear class 3 PI3K signaling. RNA-seq of WT and Vps15 liver KO at ZT6.机体代谢需求呈现节律性波动，依赖于生物钟（circadian clock）与营养感应信号通路的协同调控，但二者互作的具体机制尚未完全阐明。本研究意外发现，3型磷脂酰肌醇-3-激酶（class 3 Phosphatidylinositol-3-kinase, PI3K）——作为在自噬介导的内吞作用与溶酶体降解中发挥脂质激酶核心功能的经典蛋白——存在一项被忽视的核功能：作为异二聚体转录因子兼生物钟驱动因子BMAL1-CLOCK的共激活因子参与基因转录。3型PI3K在囊泡运输中的经典促分解代谢功能，依赖于脂质激酶Vps34与其调节亚基Vps15形成的必需复合物。本研究发现，尽管3型PI3K的两个亚基均可与RNA聚合酶II（RNA Pol II）结合，并与活跃转录位点共定位，但仅特异性缺失Vps15即可削弱BMAL1-CLOCK的转录活性。由此，我们证实了核Vps34与Vps15之间存在非冗余性：在Vps34敲除的细胞中仍存在持续存留的Vps15核池，且Vps15可独立于其与Vps34的复合物协同激活BMAL1-CLOCK。在生理状态下，Vps15对肝脏的代谢节律至关重要；且出乎意料的是，Vps15可促进促合成代谢的从头嘌呤核苷酸合成。我们证实，Vps15能够激活编码肌苷单磷酸（inosine monophosphate，嘌呤合成核心代谢中间产物）的Ppat基因的转录。最终研究表明，在抑制生物钟转录活性的禁食状态下，BMAL1在染色质上的招募并未发生改变，但此时其靶基因Nr1d1与Ppat的启动子区域的Vps15水平出现耗竭。本研究结果提示，3型磷脂酰肌醇-3-激酶的Vps15亚基核池可耦联生物钟的转录活性，实现能量稳态的时序调控，同时为解析核3型PI3K信号通路的复杂性开辟了全新研究方向。本研究包含野生型（WT）与Vps15肝脏基因敲除（KO）小鼠在昼夜节律时间点6（ZT6）的RNA测序（RNA-seq）数据。