Abstract
Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb-deficient cells using a murine intestinal model. We show that Myc and E2f1–3 have little impact on normal G1–S transitions. Instead, they synergistically control an S–G2 transcriptional program required for normal cell divisions and maintaining crypt–villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the ‘super activation’ of a G1–S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb-deficient cells hijack and redeploy Myc and E2f3 from an S–G2 program essential for normal cell cycles to a G1–S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc.
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Acknowledgements
We thank M. L. Rawahneh, N. Lovett and J. Moffitt for assistance with histology. We also acknowledge assistance from C. Martin for reviewing histological slides, S. Bae for advice on sequencing library construction, and V. Jin, Z. Ye and J. McElroy for suggestions on the analysis of sequencing data. This work was supported by the Microarray, Nucleic Acid and Analytical Cytometry Core Shared Resources at Ohio State University. We are grateful to V. Gopalan and A. Simcox for critical suggestions. This work was financially supported by NIH grants to G.L. (R01CA121275 and R01HD047470) and an NIH grant to J.M.P. (R01CA098956). H.L., T.P. and B.H. were recipients of Graduate, Postdoctoral and Undergraduate Pelotonia Fellowships, respectively.
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H.L. and G.L. designed the experiments. H.L., P.D., B.H., S.R., N.F., A.B., R.K., C.K. and M.T.S.-R. performed the experiments, collected and analysed data. X.T., A.S., T.P., K.H., R.M. and P.C. performed bioinformatic and statistical analysis for the gene expression and ChIP-exo-seq data. Z.C. and Q.W. advised for the ChIP-exo-seq experiments. J.M.P. and G.L. supervised the study. H.L. and G.L. wrote the manuscript with inputs from all authors.
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Supplementary Figure 1 Loss of E2f1-3 and/or Myc in crypts.
(a) Dual staining of EdU (red) and geminin (green, included here for the composition of merged images) in crypts from intestines harvested 2 days after induction of Ah-cre expression. Nuclei were stained with DAPI (blue). Data are representative images from n = 3 mice per genetic group. (b) Quantification of EdU and geminin staining. Data presented as mean ± s.d., n = 3 mice per genetic group. (c) IHC staining of Myc in intestines harvested 2 days after induction of Ah-cre expression. Data are representative images from n = 3 mice for each genetic group. (d) Alcian Blue staining for goblet cells (blue) and IF staining of lysozyme for paneth cells (red) in intestines harvested 2 days after induction of Ah-cre expression. Nuclei were counterstained with Nuclear Fast Red or DAPI. Data are representative images from n = 3 mice per genetic group. Scale bars in a, c and d represent 50 μm.
Supplementary Figure 2 DNA binding of E2f3 and Myc in wild type crypts.
(a) IHC staining of E2f3 using the antibody (Millipore; 05-551) in control, E2f3a−/−, E2f3b−/− and Ah-cre;E2f1−/−;E2f2−/−;E2f3loxP/loxP intestines. Data are representative images from n = 2 mice per genetic group. E2f3b−/− but not E2f3a−/− intestines show positive-stained cells, indicating the specific recognition of E2f3a isoform by this antibody. Note the non-specific staining of blood cells. (b) Heatmap representation of differentially expressed genes in wild type crypts (n = 5) versus wild type villi (n = 5). P < 0.01, empirical Bayes method. (c) ChIP-exo-seq track examples showing E2f3 and Myc binding to selected G1-S and S-G2 related genes in wild type crypts. Examples are derived from pooled crypts (n = 32 mice). Scale bars in a represent 25 μm. Scale bars in c represent 1kb.
Supplementary Figure 3 Loss of either E2f1-3 or Myc corrects aberrant transcription in Rb KO villi.
RT-qPCR analysis of mRNA levels for a subset of genes in control, Rb KO, Rb/E2f QKO and Rb/Myc DKO villi. Expression levels from individual mice are plotted (2 or 3 per genetic group as indicated) and error bars represent mean ± s.d. from n = 3 technical replicates. The aberrant expression of these genes in Rb KO villi were normalized by loss of either E2f1-3 or Myc. The average expression level of control samples was set as 1.
Supplementary Figure 4 Peak summit-distance plots and tag intensity heatmaps across tissue compartments and genetic groups.
(a) Peak summit-distance plots for E2f3 summits that are associated with the 701 dysregulated genes in Rb KO villi. E2f3 binding is compared between crypts and villi of the same genetic group. (b) Heatmap representation of tag intensity for all E2f3 binding locations. E2f3 binding is compared between crypts and villi of the same genetic group. (c) Peak summit-distance plots for E2f3 summits that are associated with the 701 dysregulated genes in Rb KO villi. E2f3 binding is compared between control and Rb KO intestines. (d) Heatmap representation of tag intensity for all E2f3 binding locations. E2f3 binding is compared between control and Rb KO intestines. (e) Peak summit-distance plots for Myc summits that are associated with the 701 dysregulated genes in Rb KO villi. Myc binding is compared between crypts and villi of the same genetic group. (f) Heatmap representation of tag intensity for all Myc binding locations. Myc binding is compared between crypts and villi of the same genetic group. (g) Peak summit-distance plots for Myc summits that are associated with the 701 dysregulated genes in Rb KO villi. Myc binding is compared between control and Rb KO intestines. (h) Heatmap representation of tag intensity for all Myc binding locations. Myc binding is compared between control and Rb KO intestines. Data in this figure (a–h) are derived from pooled crypts (n = 32 mice for control, n = 27 mice for Rb KO) or villi (n = 7 mice for control, n = 7 mice for Rb KO).
Supplementary Figure 5 E2f3 DNA binding in wild type and Rb KO villi.
(a–c) Three main categories of target genes having different patterns of E2f3 DNA binding in wild type and Rb KO villi are illustrated: Group 1 includes target genes with E2f3 peaks present in Rb KO villi but absent in control villi; Group 2 includes target genes with distinct E2f3 peaks in control and Rb KO villi (either due to additional peak summits or peak position changes); Group 3 includes target genes with E2f3 peaks similarly positioned in control and Rb KO villi (but possibly with different magnitude of binding strengths). ChIP-exo-seq track examples are shown for group 1 (a), group 2 (b) and group 3 (c). Data in this figure are derived from pooled villi (n = 7 mice for control, n = 7 mice for Rb KO). Scale bars in a–c represent 1kb.
Supplementary Figure 6 DNA binding strength and DNA binding motifs of all Myc and E2f3 peaks.
(a) Tag intensity plots (tags per bp per peak per 100M reads) around all peak summits identified in crypts and villi, as indicated. (b) Canonical DNA binding motifs (TTCCCGCC motif for E2f3, underlined with blue lines; CACGTG motif for Myc, underlined with reds lines) or strongest non-canonical motifs identified from all peak sequences in indicated genomic regions. Data in this figure are derived from pooled crypts (n = 32 mice for control, n = 27 mice for Rb KO) or villi (n = 7 mice for control, n = 7 mice for Rb KO).
Supplementary Figure 7 DNA binding motifs of Myc and E2f3 peaks associated with the 701 dysregulated genes in Rb KO villi.
Canonical DNA binding motifs (TTCCCGCC motif for E2f3, underlined with blue lines; CACGTG motif for Myc, underlined with reds lines) or strongest non-canonical motifs identified from peak sequences in indicated genomic regions of the 701 dysregulated gene in Rb KO villi. Data in this figure derived from pooled crypts (n = 32 mice for control, n = 27 mice for Rb KO) or villi of (n = 7 mice for control, n = 7 mice for Rb KO).
Supplementary Figure 8 Expression of Wnt/β-catenin targets in Rb KO villi and auto-regulation of E2fs.
(a) Venn diagram showing the overlap between the 701 genes dysregulated in Rb KO villi and the 111 Wnt/β-catenin target genes. (b) ChIP-exo-seq tracks showing E2f3 binding to the E2f1 and E2f2 loci. The differential binding between control and Rb KO villi is highlighted in blue. Examples are derived from pooled villi (n = 7 mice for control, n = 7 mice for Rb KO). Scale bars in b represent 1kb.
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Liu, H., Tang, X., Srivastava, A. et al. Redeployment of Myc and E2f1–3 drives Rb-deficient cell cycles. Nat Cell Biol 17, 1036–1048 (2015). https://doi.org/10.1038/ncb3210
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DOI: https://doi.org/10.1038/ncb3210
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