Collapse of the hepatic gene regulatory network in the absence of FoxA factors. Collapse of the hepatic gene regulatory network in the absence of FoxA factors

FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex network thought to become resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for this regulatory network, we ablated all FoxA genes in the adult liver. Remarkably, loss of FoxA caused a rapid and massive reduction in the expression of key liver genes back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining chromatin activity, nucleosome positioning and binding by other hepatic transcription factors. Thus, the hepatic gene regulatory network is dependent on the FoxA proteins throughout life. Overall design: We depleted all three FoxA genes in 8 week-old mice by injecting adeno-associated virus 8 (AAV8) carrying the gene for Cre recombinase under the control of the hepatocyte-specific thyroid-binding globulin (Tbg) promoter to adult FoxA1L/L/FoxA2 L/L/FoxA3-/- mice and validated FoxA depletion (FoxA triple null). As controls we injected the same transgenes with AAV expressing GFP. FoxA triple nulls were compared also to FoxA1/A2 lox animals, FoxA1/A2 cre alfp and to hepatoblast and new-born RNA-seq data. In addition, RNA-seq was also performed on HNF4a null livers depleted in adult livers.

FoxA转录因子（FoxA transcription factors）通过其先锋活性在肝脏发育过程中发挥关键作用：该活性可启动一套高度复杂的调控网络，该网络被认为可通过相互冗余机制，抵御任意单个肝脏转录因子缺失所带来的影响。为探究FoxA因子对该调控网络的非必需性，我们在成体小鼠的肝脏中敲除了所有FoxA基因。值得注意的是，FoxA的缺失会导致关键肝脏基因的表达快速且大规模地回落至胎儿期肝前内胚层阶段的低水平，进而引发肝细胞坏死，并在数天内导致小鼠死亡。机制研究显示，FoxA蛋白对于维持染色质活性、核小体定位以及其他肝脏转录因子的结合均不可或缺。由此可见，肝脏基因调控网络在整个生命进程中均依赖于FoxA蛋白。整体实验设计：我们向8周龄的成体FoxA1^L/L/FoxA2^L/L/FoxA3^-/-小鼠注射携带肝细胞特异性甲状腺结合球蛋白（Tbg）启动子调控下的Cre重组酶基因的8型腺相关病毒（AAV8），以敲除全部三个FoxA基因，并验证了FoxA的敲除效果（即FoxA三敲除小鼠）。作为对照，我们使用携带绿色荧光蛋白（GFP）基因的同型AAV进行注射。我们将FoxA三敲除小鼠与FoxA1/A2 lox小鼠、FoxA1/A2 cre alfp小鼠，以及肝母细胞和新生小鼠的RNA测序（RNA-seq）数据进行对比分析。此外，我们还对成体肝脏中敲除肝细胞核因子4α（HNF4α）的肝脏样本开展了RNA测序实验。