Redeployment of Myc and E2f1-3 drives Rb-deficient cell cycles

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.

Figure 1. Disruption of the small intestine by combined loss of Myc and E2f1-3

(a) Haematoxylin-and-eosin (H&E) stained tissue sections from control, E2f TKO, Myc KO and E2f/Myc QKO intestines collected 7 days after induction of Ah-cre expression. (b) Progressive degeneration of E2f/Myc QKO crypts from day 1 to day 4 after induction of Ah-cre expression. (c) H&E stained tissue sections from control and E2f/Myc QKO intestines collected 2 days after induction of Ah-cre expression. Data in a–c are representative images from n=3 mice per genetic group at each indicated time point. (d) Quantification of average number of crypt cells. Data presented as mean ± s.d., n=3 mice per genetic group at each indicated time point. (e) E2f/Myc QKO tissue sections stained by H&E and immunohistochemistry (IHC) of Myc to show the regeneration of intestinal epithelium by Myc-positive cells at 7 and 14 days after induction of Ah-cre expression (arrows). Data are representative images from n=3 mice at each time point. Scale bars represent 100 μm (a, e), 50 μm (b) and 25 μm (c).

Figure 2. S-G₂ cell cycle arrest in E2f/Myc QKO progenitor cells

(a) Immunofluorescence (IF) staining of BrdU (green), geminin (green) and P-H3 (red) 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 BrdU, geminin and P-H3 staining. Data presented as mean ± s.d., n=3 mice per genetic group. (c) Fluorescence-activated cell sorting analysis of cell cycle status in control and E2f/Myc QKO crypts from intestines harvested 2 days after induction of Ah-cre expression. Left, representative histograms from control (n=3 mice) and E2f/Myc QKO (n=5 mice) crypts. Right, quantification of histograms; data presented as mean ± s.d., n=3 (control) or n=5 (E2f/Myc QKO) mice. (d) IF staining of P-H2AX (red) and IHC staining of cleaved caspase-3 (brown) in crypts. Intestines were harvested 2 days (P-H2AX) or 4 days (cleaved caspase-3) after induction of Ah-cre expression. Data are representative images from n=3 mice per genetic group. (e) Quantification of P-H2AX and cleaved caspase-3 staining. Data presented as mean ± s.d., n=3 mice per genetic group. Scale bars in a and d represent 50 μm.

Figure 3. Synergistic regulation of an S-G₂ transcriptional program by Myc and E2f1-3

(a) Heatmap representation for clustering of differentially expressed genes between mutant genetic groups compared to control samples. Crypts were collected 2 days after induction of Ah-cre expression. n=4 for control and Myc KO mice, n=5 for E2f TKO and E2f/Myc QKO mice. P<0.05, Student’s t-test. (b) Quantitative polymerase chain reaction with reverse transcription (RT-qPCR) analysis for a subset of Group III (S-G₂ related) genes. Normal expression levels are illustrated as grey dotted lines and dysregulated expression levels in E2f/Myc QKO crypts are illustrated as red dotted lines. (c) RT-qPCR analysis for a subset of G₁-S related genes. Normal expression levels are illustrated as grey dotted lines. In b and c, expression levels from individual mice are plotted (4 per genetic group) and error bars represent mean ± s.d. from n=3 technical replicates. (d) IF staining of Pcna, Mcm3, Ccna2 and Cdc2. Note that degenerating E2f/Myc QKO crypts with less dense cells have comparable protein levels of Pcna and Mcm3, yet significantly less Ccna2 and Cdc2, compared to other genetic groups. Data are representative images from n=3 mice per genetic group. Scale bars in d represent 50 μm.

Figure 4. Chromatin binding of E2f3 and Myc in wild type tissues

(a) Heatmap of tag intensity for all E2f3 and Myc binding locations in wild type intestines. Data collected from pooled crypts (n=32 mice) or villi (n=7 mice). (b) E2f3 ChIP-PCR validation: control (Rb KO, n=3 villi) and E2f1-3 deficient (Rb/E2f QKO, n=3 villi); Myc ChIP-PCR validation: control (wild type, n=4 crypts) and Myc deficient (Myc KO, n=3 crypts). Data presented as mean ± s.d. For detailed peak location and primer sequence information see Supplementary Table 11. (c) Genomic spatial distribution of all E2f3 and Myc peak summits in wild type intestines. The number of summits in each tissue compartment is shown in parentheses. 5′ distal: 5′ region more than −50 kilobases (kb) from transcription start sites (TSSs). 5′ proximal: 5′ region within −50kb to −5kb of TSSs. Promoter: −5kb to +2kb of TSSs. Gene body: from +2kb of TSSs to end of transcripts. 3′ region: 3′ region starting from end of transcripts. (d) Density plots of all E2f3 and Myc peak summits across genomic regions in wild type intestines. Gene bodies for individual genes were divided into 100 bins and summit locations were accordingly assigned a genomic position. Data in c and d were collected from pooled crypts (n=32 mice) or villi (n=7 mice). (e) IHC staining of E2f3a and Myc in wild type intestines. Data are representative images from n=3 mice. Scale bars, 50 μm. (f) Heatmap of differential expression of cell cycle related genes, as annotated in Cyclebase database, in wild type crypts (n=5 mice) and villi (n=5 mice). P<0.01, empirical Bayes method. (g) Genomic spatial distribution of E2f3 and Myc peak summits associated with differentially expressed cell cycle related genes in wild type intestines. The data represent a subset of c and classification of genomic regions is the same as in c. (h, i) ChIP-exo-seq track examples showing E2f3 and Myc binding to selected G₁-S related genes (h) and S-G2 related genes (i) in wild type crypts. E2f3 and Myc peaks are highlighted in blue and red, respectively. Examples are derived from pooled crypts (n=32 mice). Scale bars in h and i represent 1kb.

Figure 5. Rb deficient cells require Myc to drive ectopic cell cycles

(a) H&E stained tissue sections from control, Rb KO, Rb/E2f QKO and Rb/Myc DKO intestines. Note the hyperplastic feature of Rb KO villi. (b) IF staining of BrdU and P-H3. Note the non-specific staining of blood cells in the lumen of villi. Data in a and b are representative images from n=3 mice (BrdU) or n=4 mice (H&E, P-H3) per genetic group. (c) Quantification of BrdU and P-H3 staining. Data presented as mean ± s.d., BrdU (n=3 mice), P-H3 (n=4 mice). (d) RT-qPCR analysis for indicated cyclins in control, Rb KO, Rb/E2f QKO and Rb/Myc DKO villi. Expression levels from individual animals are plotted (4 per genetic group) and error bars represent mean ± s.d. from n=3 technical replicates. (e) IHC staining of Ccna2. Data are representative images from n=3 mice per genetic group. Scale bars in a, b and e represent 50 μm.

Figure 6. Myc and E2f1-3 regulate an overlapping G₁-S transcriptional program in Rb-null cells

(a) Heatmap representation of dysregulated expression in Rb KO villi (701 genes) (left panel is from previously published data and is included here for comparison). Group IV: genes with expression levels rescued by loss of either E2f1-3 or Myc. Group V: genes with expression levels rescued by loss of E2f1-3. Group VI: genes with expression levels rescued by loss of Myc. P<0.05, Student’s t-test. (b) Waterfall plots illustrating the extent to which gene expression dysregulation in RbKO villi (701 genes) is ameliorated in Rb/E2f QKO and Rb/Myc DKO villi. (c) Venn diagram showing the overlap between genes with expression levels rescued by loss of E2f1-3 or Myc (complete and partial rescue). Data in a–c were collected from n=3 mice per genetic group for E2f-rescue experiments and n=5 mice per genetic group for Myc-rescue experiment. (d) RT-qPCR analysis for a subset of G₁-S related genes. Expression levels for individual mice are plotted (2 or 3 per genetic group as indicated) and error bars represent mean ± s.d. from n= 3 technical replicates.

Figure 7. Rb loss redefines the chromatin binding landscape of E2f3 and Myc

(a) Heatmap representation of tag intensity for all E2f3 and Myc binding locations in Rb KO intestines. Data were collected from pooled crypts (n=27 mice) or villi (n=7 mice). (b) Genomic spatial distribution of all E2f3 and Myc peak summits in control and Rb KO tissues (data for control tissues from Fig. 4c is included here for comparison). The number of summits in each tissue compartment is shown in parentheses. Data were collected 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). (c) Density plots of all E2f3 and Myc peaks across genomic regions in Rb KO crypts (pooled from n=27 mice) and villi (pooled from n=7 mice). Gene bodies for individual genes were divided into 100 bins and summit locations were accordingly assigned a genomic position. (d) Genomic spatial distribution of E2f3 and Myc peak summits associated with dysregulated genes in Rb KO villi. The number of summits in each compartment is shown in parentheses. The data represent a subset of b and classification of genomic regions is the same as in b. (e, f) Peak summit-distance plots for E2f3 summits (e) and Myc summits (f) in control crypts and Rb KO villi that are associated with the 701 dysregulated genes in Rb KO villi. (g) Heatmap representation of tag intensity for all E2f3 and Myc binding locations in control crypts and Rb KO villi. (h) Tag intensity plots (tags per bp per peak per 100M reads) around the peak summits associated with genes dysregulated in Rb KO villi. The canonical DNA binding motifs for E2f3 (TTCCCGCC) and Myc (CACGTG) are highlighted in blue and red boxes, respectively. Data in d–h were collected 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).

Figure 8. Myc regulates E2f3a expression in Rb deficient villi

(a) IHC staining of β-catenin. (b) IHC staining of Myc. Data in a and b are representative images from n=3 mice for each genetic group. (c) RT-qPCR analysis for E2f3a and E2f3b in control, Rb KO and Rb/Myc DKO villi. Expression levels for individual mice are plotted (4 per genetic group) and error bars represent mean ± s.d. from n=3 technical replicates. (d) IHC staining of E2f3a in control, Rb KO, Rb/Myc DKO and Rb/E2f QKO samples. Note the non-specific staining of blood cells in the lumen of the villi. Data are representative images from n=3 mice per genetic group. (e) ChIP-PCR analysis showing E2f3 loading to target genes (Pbk and Rrm1) in control, Rb KO and Rb/Myc DKO villi. n=4 mice per genetic group. The 5′ region ~1kb away from TSS of Gapdh was used as the negative control. *P<0.05, one-tailed Student’s t-test. (f) ChIP-exo-seq tracks showing E2f3 and Myc occupancy on the E2f3 locus. Distinct promoter regions for E2f3a and E2f3b are shaded in red and yellow, respectively. Data were collected 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). (g) RT-qPCR analysis for E2f1 and E2f2 in control and Rb KO villi. Expression levels for individual mice are plotted (4 for each genetic group) and error bars represent mean ± s.d. from n=3 technical replicates. (h) Diagrams summarizing the regulation of cell cycles by Myc and E2f1-3 in wild type and Rb deficient cells. Scale bars represent 25 μm (a) and 50 μm (b, d). Scale bars in f represent 1kb.