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Files in this Data Supplement:
Table S1. Normalized enrichment scores from GSEA. The comparison of early and late pubertal mammary gland samples through a Gene Set Enrichment Analysis (GSEA) resulted in normalized enrichment scores (NES) for a number of gene sets. The gene sets with P<0.05 are shown in the table. Positive NES indicate upregulation in the early sample, whereas negative NES reflect an upregulation in the late puberty samples. In addition, the comparison of lactating and involuting mammary gland samples through GSEA resulted in NES for a large number of gene sets. The gene sets with a false discovery rate (FDR) value of less than 0.25 are shown in the table. Positive NES indicate upregulation in the lactating sample, whereas negative NES reflect an upregulation in the involuting samples. The nominal P-value is also indicated for each gene set.
Table S2. Normalized enrichment scores from ASSESS for lactation and involution. The comparison of lactating and involuting mammary gland samples through a modified GSEA that allows individual sample variability, termed ASSESS, resulted in normalized enrichment scores (NES) for a large number of gene sets. The gene sets with at least two NES >1.4 or <−1.4 are shown in the table. Positive NES indicate upregulation in the lactating sample, whereas negative NES reflect an upregulation in the involuting samples.
Table S3. Normalized enrichment scores from ASSESS for the puberty dataset. The comparison of early and late puberty through ASSESS resulted in normalized enrichment scores (NES) for a large number of gene sets. The gene sets having at least two NES >1.4 or <−1.4 are shown for this group. Positive NES are indicative of upregulation during early puberty, whereas negative NES are characteristic of upregulation in late puberty.
Table S4. Transcription factor binding. Genes that were elevated 2-fold on involution day 1 in wild-type or E2f3 heterozygous mammary glands were used in GATHER to search for consensus promoter binding sites using TRANSFAC. For both wild-type and E2f3 heterozygous samples, the top 15 promoter binding sites, the total number of genes in each promoter consensus site group and various measures of statistical significance are shown. The various genes in the E2F groups were combined and are shown below the table for both involution day 1 and involution day 2. Six target genes that were identified as outlined above were tested for E2F3 binding. The number of E2F consensus promoter binding sites is shown for each of the six targets that were examined. The fold elevation represented graphically in Fig. S6 (see Fig. S6 in the supplementary material) is listed for all of the targets, as well as the ribonucleotide reductase M2 (RR2) control for comparison of a well-established target. The P-value shown depicts the significance of enrichment of the E2F3 immunoprecipitation over the normal rabbit control immunoprecipitation. Fold elevation is relative to albumin controls.
Fig. S1. ASSESS results. Complete results for ASSESS comparing lactation and involution. All gene sets with a normalized enrichment score greater than 1.4 in at least two samples were clustered and are displayed.
Fig. S2. ASSESS results for early versus late puberty. Complete results for ASSESS comparing early and late puberty. All gene sets with a normalized enrichment score greater than 1.4 in at least two samples were clustered and are displayed.
Fig. S3. Stat3 and E2F expression profile. (A) Average gene expression levels from microarray analysis for each of the two probes for Stat3 are shown for the mammary development dataset. The standard deviation is indicated from the three samples for each of the probes. (B) Through quantitative RT-PCR, E2F expression was analyzed in various tissues. Results were standardized against bone marrow to assess relative levels. (C) E2F expression in the mammary gland was assessed at various developmental stages by quantitative RT-PCR and was standardized against the adult mammary gland. (D) To further examine the E2F3 and E2F4 expression patterns, wild-type sections from lactation and involution were examined through immunohistochemistry for E2F3 and E2F4.
Fig. S4. TEB/stroma fold change. The top 200 genes as measured by fold change when comparing TEBs to ducts. In addition, these genes were examined through GATHER to assess which transcription factor binding sites they contained. From the top 20 results in GATHER for transcription factor binding, those genes with an E2F-binding site are listed.
Fig. S5. Involution signature generation and prediction. (A-C) We used supervised classification methods to derive involution signatures comparing involution day 1 with day 2, 3 or 4. The image intensity display of the expression level of genes used in the predictor to differentiate between the developmental stages is shown with columns reflecting samples and rows reflecting genes. (D-F) Using binary regression analysis, the probability of the wild-type or E2f3 mutant samples matching the three predictors (A-C) is shown with high probability (red) to low probability (blue). (G-I) These predictions are also shown in plots, where P-values for t-tests for the differences between wild-type and E2f3 mutant samples are shown.
Fig. S6. E2F3 target validation by chromatin immunoprecipitation. For several targets identified as being potentially regulated by E2F3 (see Table S4 in the supplementary material), we tested the ability of E2F3 to bind their promoters in a chromatin immunoprecipitation (ChIP) assay in both asynchronous proliferating HC11 cells and in HC11 cells undergoing apoptosis. For the genes with a significant enrichment of the target in the E2F3 immunoprecipitated sample relative to the normal rabbit (NR) sample, a plot of their normalized expression levels from an involution timecourse is also shown. Ribonucleotide reductase M2 (RR2), a well-established E2F3 target, is included as a positive control and albumin is included as a negative control. For complete ChIP results and P-values, see Table S4 in the supplementary material.
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