Transcription factor compensation during mammary gland development in E2F knockout mice

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.