Coordinated Cross-Talk Between the Myc and Mlx Networks in Liver Regeneration and Neoplasia

The Myc bHLH-ZIP transcription factor is deregulated by most cancers. As a heterodimer with the bHLH-ZIP protein Max, Myc regulates target genes that contribute to metabolism and proliferation. This “Myc Network” cross-talks with the “Mlx Network” comprised of the Myc-like bHLH-ZIP proteins MondoA and ChREBP and the Max-like bHLH-ZIP protein Mlx. This “Extended Myc Network” regulates genes with both common and distinct functions. We have generated hepatocytes lacking Mlx (mlxKO) or Mlx+Myc (double KO or DKO) and quantified their abilities to replace dying hepatocytes in a murine model of Type I tyosinemia. We find that this function deteriorates as the Extended Myc Network is progressively dismantled. Genes dysregulated in mlxKO and DKO hepatocytes include those involved in translation and mitochondrial function. The Myc and Mlx Networks thus cross-talk with the latter playing a disproportionate role. mycKO and mlxKO mice also develop age-dependent non-alcoholic fatty liver disease and mlxKO and DKO mice develop extensive hepatic adenomatosis not observed in wild-type, mycKO, chrebpKO or mycKOxchrebpKO mice. In addition to demonstrating cooperation between the Myc and Mlx Networks, this study reveals the latter to be more important in maintaining metabolic and translational homeostasis, while concurrently serving as a suppressor of benign tumorigenesis. Overall design: RNA purification from four to six representative livers of each group was performed using Qiagen RNAeasy columns (Qiagen, Inc., Valencia, CA) followed by DNase digestion. Sample integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). All samples had RIN values of 8.5-10 prior to any further processing. Sequencing was performed on a NovaSeq 600 Instrument (Illumina, Inc., San Diego, CA) by Novagene, Inc. (Sacramento, CA).

Myc碱性螺旋-环-螺旋亮氨酸拉链（bHLH-ZIP）转录因子在多数癌症中发生失调。作为与bHLH-ZIP蛋白Max形成的异二聚体，Myc可调控参与代谢与细胞增殖的靶基因。该"Myc网络"可与由类Myc bHLH-ZIP蛋白MondoA、ChREBP以及类Max bHLH-ZIP蛋白Mlx组成的"Mlx网络"发生信号串扰。这个"扩展Myc网络"可调控兼具共同与独特功能的靶基因。我们构建了缺失Mlx的肝细胞（mlxKO）或同时缺失Mlx与Myc的肝细胞（双敲除，DKO），并在I型酪氨酸血症小鼠模型中量化其替代死亡肝细胞的能力。研究发现，随着扩展Myc网络被逐步拆解，这一代偿功能逐渐受损。在mlxKO与DKO肝细胞中失调的基因涵盖参与翻译过程与线粒体功能的相关基因。由此可见，Myc网络与Mlx网络存在信号串扰，且后者发挥了更为关键的调控作用。mycKO与mlxKO小鼠还会出现年龄依赖性非酒精性脂肪性肝病；而mlxKO与DKO小鼠会出现广泛的肝腺瘤病，该表型在野生型、mycKO、chrebpKO或mycKOxchrebpKO小鼠中均未观察到。本研究不仅证实了Myc网络与Mlx网络间的协同作用，还揭示后者在维持代谢与翻译稳态中更为核心，同时可作为良性肿瘤发生的抑制因子。整体实验设计：使用Qiagen RNAeasy柱式试剂盒（Qiagen公司，美国加利福尼亚州巴伦西亚）从每组4至6只代表性小鼠的肝脏中提取总RNA，随后进行DNase消化处理。采用Agilent 2100生物分析仪（Agilent Technologies公司，美国加利福尼亚州福斯特城）检测样本完整性，所有样本在进一步处理前的RNA完整性数值（RIN）均处于8.5至10之间。测序工作由Novagene公司（美国加利福尼亚州萨克拉门托）在NovaSeq 600测序仪（Illumina公司，美国加利福尼亚州圣地亚哥）上完成。