Fig 1.
E2F transcription factors define mammary developmental states.
Here we have combined a shRNA screen in primary mouse MaSC-enriched basal cells[5] with E2F1-3 gene expression signatures [25] to define progenitor genes regulated by the E2F transcription factors (A). Using a Venn diagram, overlap between E2F1, E2F2 and E2F3 target genes as determined by CHIP-seq/CHIP-chip and microarray experiments, and the 73 unique genes identified from the pooled shRNA screen was noted (B). Unsupervised hierarchical clustering of E2F1 signature genes from mammary glands collected at various stages of mammary development is stratified into the indicated stages. The overlap between E2F1, E2F2 and E2F3 signature genes is depicted on the right of the of the heatmap (C).
Fig 2.
E2F regulated genes in the terminal end bud.
Combining the E2F1, E2F2 and E2F3 signature genes and differentially expressed genes derived from microarray with a TEB / duct dataset [4] revealed E2F regulated genes in the TEB. Further, E2F ChIP-seq and ChIP-chip data was combined with this TEB/duct dataset to predict direct E2F targets in the TEB (A). Using Venn Diagrams to compare TEB E2F1, E2F2 and E2F3 signature genes/differentially expressed genes revealed which genes were regulated singly and by multiple E2F transcription factors (B). Similarly, direct TEB E2F1, E2F2 and E2F3 targets were analyzed (C).
Fig 3.
Progenitor genes, mammary stages and transcription factor enrichment.
Unsupervised hierarchical clustering of 17 mammary gland developmental timepoints using genes with a potential role in mammary gland development as determined by the pooled shRNA screen. Each of the 17 developmental timepoints have three replicates. The overlap between potential mammary stem/progenitor cell genes and E2F1 direct and predicted target genes as determined by ChiP-ChIP and GATHER is depicted on the right side of the heatmap. The predicted E2F1 targets have a p-value of 0.002 and a Bayes factor of 3. (A). Examining transcription factors with overlap of ChIP data in the various stages of mammary development highlights the importance of these genes in mammary gland function. The E2F1 transcription factor is highlighted (yellow) with over 30 of the 73 genes being regulated (B).
Fig 4.
Compensation during mammary gland function.
The levels of E2F1, E2F2, E2F3a and E2F3b were quantified in mammary gland RNA at pregnancy day 14.5 in wild type (WT) FVB (n = 3), E2F1-/- (n = 3), E2F2-/- (n = 3) and E2F3+/- (n = 3) mice. A significant increase in E2F2 and E2F3a was observed in E2F1-/- mice. A significant increase in E2F2 was also observed in E2F3+/- mice (A). The levels of E2F1, E2F2, E2F3a and E2F3b were quantified at lactation day 5 in WT FVB (n = 3), E2F1-/- (n = 3), E2F2-/- (n = 3) and E2F3+/- (n = 3) mice. A significant increase in E2F1 and E2F3b was observed in the E2F2 null mice. A significant increase in E2F2 was also noted in E2F3 heterozygous knockout mice (B). This analysis revealed differential E2F specific compensatory gene expression dependent upon the developmental context of the mammary gland. A significant increase in E2F expression levels relative to WT control mice are depicted with an asterisk. * p value < 0.05 and ** p value < 0.006.
Fig 5.
Mammary gland outgrowth in double knockouts.
Representative wholemount images of mammary gland outgrowth at 4 weeks of development in wild type controls (A), E2F1-/- (B), E2F2-/- (C), E2F1-/-/E2F2-/- (D) E2F2-/-/E2F3+/- (E), E2F1-/-/E2F3+/- (F) mice are shown. Measuring from nipple to lymph node (LN) as well as from nipple to the most distal TEB (arrowhead) allowed for quantification of mammary gland outgrowth. The results of the E2F1/E2F2 (E), E2F1/E2F3 (F) and E2F2/E2F3 (G) mammary outgrowth experiments are shown as a percentage of the wild type control growth. The mammary outgrowth was quantified in 12 WT FVB, 11 E2F1-/-, 15 E2F2-/-, 8 E2F3+/-, 5 E2F1-/-/E2F2-/-, 5 E2F1-/-/E2F3+/- and 3 E2F2-/-/E2F3+/-. There is a significantly greater delay in mammary outgrowth in the E2F1-/-/E2F2-/- mice relative to the E2F1-/- mice. Differences in mammary outgrowth delay that are significant between two strains are depicted with an asterisk. * p value < 0.05 and ** p value < 0.006.