Department of Physiology, Michigan State University, East Lansing, MI, United States of America.
PLoS One. 2018 Apr 4;13(4):e0194937. doi: 10.1371/journal.pone.0194937. eCollection 2018.
The E2F transcription factors control key elements of development, including mammary gland branching morphogenesis, with several E2Fs playing essential roles. Additional prior data has demonstrated that loss of individual E2Fs can be compensated by other E2F family members, but this has not been tested in a mammary gland developmental context. Here we have explored the role of the E2Fs and their ability to functionally compensate for each other during mammary gland development. Using gene expression from terminal end buds and chromatin immunoprecipitation data for E2F1, E2F2 and E2F3, we noted both overlapping and unique mammary development genes regulated by each of the E2Fs. Based on our computational findings and the fact that E2Fs share a common binding motif, we hypothesized that E2F transcription factors would compensate for each other during mammary development and function. To test this hypothesis, we generated RNA from E2F1-/-, E2F2-/- and E2F3+/- mouse mammary glands. QRT-PCR on mammary glands during pregnancy demonstrated increases in E2F2 and E2F3a in the E2F1-/- mice and an increase in E2F2 levels in E2F3+/- mice. During lactation we noted that E2F3b transcript levels were increased in the E2F2-/- mice. Given that E2Fs have previously been noted to have the most striking effects on development during puberty, we hypothesized that loss of individual E2Fs would be compensated for at that time. Double mutant mice were generated and compared with the single knockouts. Loss of both E2F1 and E2F2 revealed a more striking phenotype than either knockout alone, indicating that E2F2 was compensating for E2F1 loss. Interestingly, while E2F2 was not able to functionally compensate for E2F3+/- during mammary outgrowth, increased E2F2 expression was observed in E2F3+/- mammary glands during pregnancy day 14.5 and lactation day 5. Together, these findings illustrate the specificity of E2F family members to compensate during development of the mammary gland.
E2F 转录因子控制着包括乳腺分支形态发生在内的发育的关键要素，其中几个 E2F 发挥着至关重要的作用。先前的其他数据表明，单个 E2F 的缺失可以被其他 E2F 家族成员所补偿，但这尚未在乳腺发育的背景下进行测试。在这里，我们探索了 E2F 及其在乳腺发育过程中相互功能补偿的作用。利用末端芽的基因表达和 E2F1、E2F2 和 E2F3 的染色质免疫沉淀数据，我们注意到每个 E2F 调控的乳腺发育基因既有重叠又有独特。基于我们的计算发现以及 E2F 共享一个共同的结合基序的事实，我们假设 E2F 转录因子在乳腺发育和功能过程中会相互补偿。为了验证这一假设，我们从 E2F1-/-、E2F2-/-和 E2F3+/- 小鼠的乳腺中提取 RNA。在怀孕期的乳腺中进行 QRT-PCR 表明，E2F1-/- 小鼠中 E2F2 和 E2F3a 的表达增加，E2F3+/- 小鼠中 E2F2 水平增加。在哺乳期，我们注意到 E2F2-/- 小鼠中 E2F3b 转录水平增加。鉴于之前已经注意到 E2F 在青春期发育过程中对发育有最显著的影响，我们假设在那个时候单个 E2F 的缺失会得到补偿。生成了双突变小鼠并与单敲除小鼠进行了比较。与单独敲除相比，E2F1 和 E2F2 的缺失显示出更明显的表型，表明 E2F2 补偿了 E2F1 的缺失。有趣的是，虽然 E2F2 在乳腺生长过程中不能对 E2F3+/- 进行功能补偿，但在怀孕第 14.5 天和哺乳期第 5 天，E2F3+/- 乳腺中观察到 E2F2 表达增加。总之，这些发现说明了 E2F 家族成员在乳腺发育过程中补偿的特异性。
