Mammary Alveolar Proliferation and Differentiation During Early Pregnancy is Regulated by E2F5

Abstract The E2F transcription factors are well-established cell cycle regulators, but their roles in coordinating proliferation and differentiation remain poorly understood. Here, we investigated the function of E2F5 during mammary gland development using a mammary epithelial-specific conditional knockout model. We found that E2F5 expression and chromatin binding increase during early pregnancy, coinciding with the critical window of alveolar development. Loss of E2F5 resulted in delayed alveolar expansion during early pregnancy, characterized by reduced side branching and smaller alveolar structures during early pregnancy. Mechanistically, E2F5 deletion led to reduced expression of canonical E2F target genes involved in cell cycle progression. Surprisingly, E2F5 loss also caused enrichment of luminal progenitor populations at the expense of differentiated alveolar cells, with chromatin profiling revealing substantial depletion of the repressive H3K27me3 mark at luminal progenitor-associated genes. These findings suggest that E2F5 promotes differentiation of luminal progenitors into proliferative alveolar precursors during early pregnancy. We propose that E2F5 coordinates both proliferation and differentiation by driving progenitor cells to differentiate into mature luminal cells. The dual function of E2F5 in mammary development distinguishes it from classical cell cycle regulators and positions it as a critical coordinator of the developmental transitions required for lactation. Competing Interest Statement The authors have declared no competing interest. Footnotes This work was supported by a grant from the NICHD 1R01HD104606-01 to ERA and a Susan Komen ASPIRE grant to JGL. Added confocal data, chromatin profiling and integration of RNAseq with the new CUT&RUN data to examine luminal progenitor differentiation.