Mammary gland development and function requires precise stage and spatial control of transcriptional programs. These processes are tightly controlled to ensure the successful function of the mammary gland, including lactation which is essential for rearing of offspring. Recent work has demonstrated a role for the E2F transcription factors that goes well beyond their traditional role in cell cycle regulation. The role of the activator E2Fs (E2F1-3a) is well characterized, including regulation of mammary gland development. However, little is known of the role of the repressor E2Fs, E2F4 and E2F5, in the mammary gland and this is partially due to hydrocephaly and early lethality in the knockout strains. The long term goal of our work is to define the role of transcriptional regulation in mammary gland development and function. Understanding normal biology then informs our studies of how this goes awry and results in breast cancer. The immediate objective of this proposal, which is the next step in our long-term goal, is to precisely define the role of repressor E2Fs in the mammary gland. Our central hypothesis is that the repressor E2Fs regulate key mammary gland developmental genes. This hypothesis was based on preliminary data from knockout mice. Indeed, loss of E2F5 in the mammary epithelium resulted in delayed ductal extension during puberty, delayed involution and alveolar overgrowth in virgin adult mice. Loss of E2F4 also resulted in delayed ductal outgrowth but was also associated with a profound lack of alveolar expansion during pregnancy and lactation. Combining ChIP-Seq data and our gene expression data, we predicted both unique and shared E2F target genes with mammary development roles. The rationale for the proposed work is that once we have completed this proposal, we will understand how transcriptional repression regulates development of the mammary gland. We plan to test our central hypothesis and accomplish the objective of this application by investigating the following specific aims. In the first aim we will characterize the mammary gland phenotypes associated with the mammary epithelial cell specific knockout of E2F5. In the second aim we will generate ChIP-Seq data and integrate it with E2F5 induced gene expression data to determine E2F5 targets. Target genes will be filtered and prioritized using additional datasets describing mammary development. Finally, in the third aim we will examine scRNAseq data in virgin glands lacking E2F3 or E2F5. This proposal is innovative because it will elucidate the genetic mechanisms regulating mammary gland development and function by the repressor E2F transcription factors. This contribution is significant because it will establish a role for E2F5 in mammary gland development.