Biology Department, California State University Channel Islands, Camarillo, California, United States of America.
The Keiki Kohola Project, Lahaina, Hawaii, United States of America.
PLoS One. 2023 Aug 29;18(8):e0284834. doi: 10.1371/journal.pone.0284834. eCollection 2023.
Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke the induction of gene expression by exercise, hypoxia, and other physiological gene regulatory pathways. Here we explore an alternative hypothesis: that cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, shows a low level of expression, only about 8% that of humans. This low expression level is broadly shared among cetaceans and artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5' of the AT element termed the "CCAC-box". Analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3' of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5' of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. We discuss the possibility that low transcription levels are actually beneficial, and that evolution of the myoglobin protein toward enhanced stability is a critical factor in the accumulation of high myoglobin levels in adult cetacean muscle tissue.
鲸类和其他潜水哺乳动物已经适应了水生环境，其中包括骨骼肌中高水平的携氧细胞内血红蛋白肌红蛋白。关于导致这些高肌红蛋白水平的机制的假说，通常涉及运动、缺氧和其他生理基因调控途径诱导基因表达。在这里，我们探索了一个替代假说：鲸类肌红蛋白基因通过驱动肌肉细胞分化的内在发育机制，进化出了高水平的转录。我们使用分化的 C2C12 细胞中的荧光素酶测定来检验这一假说。与我们的假设相反，我们发现，来自小须鲸（Balaenoptera acutorostrata）的肌红蛋白基因表达水平很低，仅为人的 8%左右。这种低表达水平在鲸类和偶蹄目动物中广泛存在。以前关于人类基因调控的研究已经确定了一个由两个区域组成的核心肌肉特异性增强子，即“AT 元件”和 AT 元件 5'端的富含 C 的序列，称为“CCAC 盒”。对小须鲸基因的分析支持 AT 元件的重要性，但小须鲸 CCAC 盒同源物的作用很小。相反，在 AT 元件 3'端的一个富含 G 的区域中发现了关键的正输入。此外，尽管在人类基因中被指定为抑制作用，但外显子 1 中的保守 E 盒也会对表达产生积极影响。最后，还鉴定出了一个位于核心增强子 5'端的新区域，我们假设该区域可能作为边界元件发挥作用。这些结果说明了进化过程中的调控灵活性。我们讨论了低转录水平实际上可能是有益的，以及肌红蛋白蛋白向增强稳定性的进化是导致成年鲸类肌肉组织中肌红蛋白水平升高的关键因素的可能性。
