Activation of the aryl hydrocarbon receptor by dioxin during embryonic stem cell differentiation disrupts the expression of homeobox transcription factors that control cardiomyogenesis

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that regulates the expression of xenobiotic detoxification genes and is a critical mediator of gene-environment interactions. In addition, many AHR target genes that have been identified by genome-wide profiling have morphogenetic functions, suggesting that AHR activation may play a role in embryonic development. To address this hypothesis, we studied the consequences of AHR activation by TCDD, its prototypical ligand, during spontaneous mouse ES cell differentiation into contractile cardiomyocytes. Treatment with TCDD or shRNA-mediated AHR knockdown significantly decreased the ability of cardiomyocytes to contract and the expression of cardiac markers in these cells. An AHR-positive embryonic stem cell lineage was generated that expressed puromycin resistance and eGFP under the control of the AHR-responsive Cyp1a1 promoter. Cells of this lineage were over 90% pure and expressed AHR as well as cardiomyocyte markers. Analysis of temporal trajectories of global gene expression in these cells shows that activation of the AHR/TCDD axis disrupts the concerted expression of genes that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation, including dozens of genes encoding homeobox transcription factors and Polycomb and Trithorax Group genes. More than 50% of the homeobox factors so regulated do not have AhRE sites in their promoters, indicating that AHR activation may establish a complex regulatory network that reaches beyond direct AHR signaling and is capable of disrupting various aspects of embryonic development, including cardiomyocyte differentiation.

芳香烃受体（aryl hydrocarbon receptor, AHR）是一种配体激活的转录因子，可调控异源生物解毒基因的表达，是基因-环境互作的关键介导因子。此外，通过全基因组谱分析鉴定出的诸多AHR靶基因具有形态发生功能，这提示AHR激活可能在胚胎发育中发挥作用。为验证这一假说，我们探究了其经典配体TCDD激活AHR后，对小鼠胚胎干细胞自发分化为收缩型心肌细胞过程的影响。TCDD处理或短发夹RNA（short hairpin RNA, shRNA）介导的AHR基因敲低，均会显著降低心肌细胞的收缩能力以及此类细胞中心肌标志物的表达水平。我们构建了一种AHR阳性的胚胎干细胞谱系，该谱系在受AHR调控的Cyp1a1启动子驱动下表达嘌呤霉素抗性基因与增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）。该谱系的细胞纯度可达90%以上，同时表达AHR与心肌标志物。对这些细胞的全局基因表达时间轨迹进行分析后发现，AHR/TCDD信号轴的激活会破坏调控心脏与神经形态发生及分化相关多条信号通路的基因的协同表达，其中包括数十个编码同源框转录因子的基因，以及多梳家族（Polycomb）和三胸群（Trithorax Group）基因。在上述受调控的同源框因子中，超过50%的基因启动子区域不含有AHR应答元件（aryl hydrocarbon receptor response elements, AhRE）位点，这表明AHR激活可能构建出一套超越直接AHR信号调控的复杂调控网络，并能够干扰胚胎发育的多个环节，包括心肌细胞分化。