Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish

Background: In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (non-functionalization), acquire a new function (neo-functionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (sub-functionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter. A previous report showed that zebrafish fabp1b.1 and fabp1b.2 promoters underwent sub-functionalization of transcriptional control by peroxisome proliferator-activated receptors (PPAR). The fabp1b.1 promoter retained a functional PPAR response element (PPRE), while fabp1b.2 did not.
Results: In this study, we describe the divergent PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters from the ancestral fabp1 gene. Promoter evolution was assessed by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine ex vivo cells, and in HEK293 cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. Spotted gar fabp1, representative of the ancestral fabp1, was responsive to both PPARα and PPARγ, and displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, while fabp1b.2 was not regulated by PPAR.
Conclusions: The zebrafish fabp1 promoters underwent two successive rounds of PPAR-selective subfunctionalization leading to retention of three zebrafish fabp1 genes that display stimuli-specific regulation.

背景：在重复-退化-互补（DDC）模型中，重复基因存在三种潜在命运：可通过突变积累丧失功能（非功能化）、获得全新功能（新功能化），或是每个重复基因保留祖先基因的部分功能（亚功能化）。目前学界对于启动子进化在真核生物基因组重复基因保留过程中所发挥的作用尚不明晰。脂肪酸结合蛋白（Fabp）属于序列与功能高度保守的多基因家族，但其基因调控模式存在差异，这提示选择压力通过启动子中的调控元件施加。既往研究显示，斑马鱼fabp1b.1与fabp1b.2启动子发生了由过氧化物酶体增殖物激活受体（PPAR）介导的转录调控亚功能化：fabp1b.1启动子保留了功能性PPAR反应元件（PPRE），而fabp1b.2则未保留该元件。
结果：本研究针对源自祖先fabp1基因的斑马鱼fabp1a、fabp1b.1与fabp1b.2启动子，阐明了其分化的PPAR调控模式。研究通过序列分析评估启动子进化情况，并在斑马鱼肝脏与肠道离体培养细胞、转染野生型及突变型fabp1启动子-报告基因构建体的HEK293细胞中，检测了不同PPAR激动剂对fabp1启动子活性的激活差异。结果显示，代表祖先fabp1基因的斑点雀鳝fabp1对PPARα与PPARγ均有响应，且对PPARα激活呈现双相响应；斑马鱼fabp1a表现为PPARα选择性，fabp1b.1为PPARγ选择性，而fabp1b.2则不受PPAR调控。
结论：斑马鱼fabp1启动子经历了两轮连续的PPAR选择性亚功能化过程，最终保留了三个呈现刺激特异性调控模式的斑马鱼fabp1基因。