Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development - PubMed (original) (raw)

doi: 10.1016/j.devcel.2007.10.017.

Cong Ran, Edward Li, Faye Gordon, Grant Comstock, Hasan Siddiqui, Whitney Cleghorn, Hui-Zi Chen, Karl Kornacker, Chang-Gong Liu, Shusil K Pandit, Mehrbod Khanizadeh, Michael Weinstein, Gustavo Leone, Alain de Bruin

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Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development

Jing Li et al. Dev Cell. 2008 Jan.

Abstract

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.

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Figures

Figure 1

Figure 1

Generation of E2f7 and E2f8 knockout mice. (A) Targeting strategies used to inactivate E2f7 (left) and E2f8 (right). Top panels: partial exon/intron structures of the E2f7 and E2f8 genes. The black bars indicate the position of probes used for Southern analysis. Middle panels (targeting vectors): position of the TK and Neo cassettes, as well as loxP sites (filled triangles) are indicated. Bottom two panels (conditional knockout alleles and conventional knockout alleles): illustrate the two desired alleles resulting from possible recombination events. (B) Top panels: Southern analysis of genomic DNA isolated from conventional knockout mice using AseI for E2f7 (left) and EcoRV for E2f8 (right), and hybridized using probes A and B, respectively. Bottom panels: genotyping of tail DNA was performed using allele-specific PCR primers. (C) Real-time RT-PCR analysis of E2f7 or E2f8 expression in embryos with the indicated genotypes using primers described in Figure S7. (D) Wild type E9.5 embryos were subjected to whole mount in situ hybridization using sense and antisense probes specific to E2f7 (top panels) and E2f8 (bottom panels).

Figure 2

Figure 2

Global deletion of E2f7 and E2f8 results in developmental delay, vascular defects and widespread apoptosis in vivo. (A) Micrograph of E2f7+/-E2f8+/-, E2f7+/-_E2f8_-/- and _E2f7_-/-E2f8+/- mice at 21 days of age. (B) Tabulated number of E2f7+/-_E2f8_-/- and _E2f7_-/-E2f8+/- mice that live until 30 (P30) and 90 (P90) days of age. (C) Gross pictures of E2f7 and E2f8 double knockout embryos at E9.5 (top panels) and E10.5 (bottom panels). The right panel is a higher magnification view of the vascular defects in E10.5 _E2f7_-/-_E2f8_-/- embryos. (D) E9.5 embryos with the indicated genotypes were analyzed by TUNEL assays. Far left panels: low magnification pictures of whole embryos. Right two panels: high magnification images of representative areas demarcated by the box in the low magnification images. (E) Quantification of TUNEL-positive cells in the indicated tissue areas are presented as the average ± SD percentage of cells that are TUNEL-positive. Three sections per embryo and at least three different embryos for each genotype were analyzed.

Figure 3

Figure 3

E2F7 and E2F8 homo- and hetero-dimerize. (A) Homo-dimerization of E2F7. Lysates from cells transfected with both flag-tagged E2F7 (flag-7) and HA-tagged E2F7 (HA-7) were immunoprecipitated (IP) and immunoblotted (IB) with anti-flag and anti-HA antibodies as indicated. Immunoprecipitation with normal mouse IgG was used as a negative control. (B) Left panels: stable expression of flag-HA-E2F7 in HeLa cells. Flag-HA-E2F7 transduced HeLa cell lysates were subjected to Western blotting. The presence of a non-specific protein indicated by (**) was used as a loading control. Right panels: Endogenous E2F8 associates with flag-HA-E2F7. Nuclear extracts from flag-HA-E2F7 expressing cells were immunoprecipitated with flag antibody affinity resins as described in Experimental Procedures and eluents were immunoblotted with E2F8-specific antibodies. Mock-transduced HeLa cells (con.) were used as a negative control. (C) Hetero-dimerization of E2F7 and E2F8. Lysates derived from cells overexpressing both flag-E2F7 and HA-E2F8 were immunoprecipitated (IP) with anti-flag antibodies and immunoblotted (IB) with anti-HA antibodies (top panel) or vice versa (bottom panel). Immunoprecipitation with normal mouse IgG was used as a negative control. (D) Kinetic analysis of dimerization. HEK 293 cells overexpressing the indicated constructs were subjected to anti-flag immunoprecipitation (IP), followed by anti-HA or anti-myc immunoblotting (IB) as indicated. The amount of detected E2F7 and E2F8 was measured by densitometry and quantified relative to 1% of the input material. The relative levels indicated at the bottom of each lane are presented as the average ±SD of 3 independent experiments where Input is always equal to 1.00. (E) Bar graph presentation of the kinetic analysis of dimerization shown in (D). (F) E2F7 binds to the E2f1 promoter. Chromatin from cells overexpressing wild type flag-E2F7 (wt) or flag-E2F7DBD1,2 (mut) was immunoprecipitated (IP) with anti-flag or IgG control antibodies. Immunoprecipitated DNA was amplified using primers specific for the E2f1 promoter (E2f1), irrelevant sequences in exon 1 of E2f1 and the tubulin promoter (control and tubulin, respectively). (G) E2F8 binds to the E2f1 promoter. Cells overexpressing wild type flag-E2F8 (wt) or flag-E2F8DBD1,2 (mut) was immunoprecipitated (IP) with anti-flag or normal mouse IgG antibodies. Immunoprecipitated DNA was amplified using primers specific for the E2f1 promoter (E2f1), exon 1 of E2f1 (control) and the tubulin promoter (tubulin). (H-J) Homo-dimers and hetero-dimers of E2F7 and E2F8 occupy the E2f1 promoter. Cell extracts expressing ectopic HA-E2F7 and flag-E2F7 (H), HA-E2F8 and flag-E2F8 (I), HA-E2F7 and flag-E2F8 (K) were used for sequential ChIP assays as described in Experimental Procedures. Antibodies used for the first and second round of immunoprecipitation are indicated (1st IP and 2nd IP, respectively). Immunoprecipitated DNA collected after two rounds of ChIP was amplified using primers specific for the E2f1 promoter (E2f1) or for the tubulin promoter (tubulin). For all the ChIP experiments, real-time PCR was performed in triplicate and cycle numbers were normalized to 1% of the input DNA.

Figure 4

Figure 4

(A-F) Deregulation of E2f1 and p53 expression in MEFs deficient in E2f7 and E2f8. (A) Top panels: PCR genotyping analysis of DNA isolated from E2f7+/+E2f8+/+ and E2f7loxP/loxPE2f8loxP/loxP MEFs treated with _cre_-expressing retroviruses. The knockout (ko) and wild type (wt) alleles are indicated. Bottom graphs: Real-time RT-PCR analysis of E2f7 and E2f8 expression in cells. For convenience, cre-deleted loxP alleles were labeled as (-/-) at bottom of graphs. (B) Western blot analysis of lysates described in (A) using antibodies specific for E2F1 and p53 as indicated. Tubulin-specific antibodies were used as an internal loading control. (C) Expression of E2f1 in MEFs treated as in (A) was measured by real-time RT-PCR. (D) Cells treated as in (A) were co-transfected with the E2f1 firefly luciferase and thymidine kinase renilla luciferase reporter plasmids (internal control). Relative E2f1-luc reporter activity was normalized to renilla luciferase activity. A representative of at least three independent experiments is shown. (E) FACS analysis of synchronized cre-treated E2f7+/+E2f8+/+ and E2f7loxP/loxPE2f8loxP/loxP MEFs. For convenience, cre-deleted loxP alleles were labeled as (-/-). MEFs were synchronized by serum deprivation and hydroxyurea (HU) block as described in Experimental Procedures. At the indicated time points, cells were harvested and stained by propidium iodide for flow cytometry. Q: quiescent cells kept in serum-deprived medium. (F) Total RNA isolated from same synchronized MEFs samples as in (E) was analyzed by real-time RT-PCR assays specific for E2f1 expression. (G-I) MEFs deficient in E2f7 and E2f8 are hypersensitive to DNA damage induced apoptosis. (G) Cre-treated E2f7+/+E2f8+/+ and E2f7loxP/loxPE2f8loxP/loxP MEFs were mock treated with DMSO (-camptothecin) or with camptothecin (+camptothecin). At the indicated intervals cells were harvested and counted in triplicate as described in Experimental Procedures. The percentages of viable cells at the indicated time points are presented. (H) Lysates derived from MEFs treated as in (G) were analyzed at indicated time points by Western blotting using caspase-3 specific antibodies. (I) Total RNA was extracted from cells treated for 18h as in (G) and expression of the indicated p53 target genes was measured by real-time RT-PCR.

Figure 5

Figure 5

Loss of E2f1 or p53 suppresses apoptosis in _E2f7_-/-_E2f8_-/- embryos. (A) Micrographs of TUNEL stained E9.5 _E2f7_-/-_E2f8_-/-_E2f1_-/- (top panels) and _E2f7_-/-_E2f8_-/-_p53_-/- embryos (bottom panels). Far left panels: low magnification pictures of whole embryos. Right three panels: high magnification images of representative areas demarcated by the box in the low magnification images. (B) Quantification of TUNEL-positive cells in the indicated tissue areas are presented as the average ± SD percentage of cells that are TUNEL-positive. Three sections per embryo and at least three different embryos for each genotype were analyzed. For comparison purposes, data derived from E2f7+/-E2f8+/- and _E2f7_-/-_E2f8_-/- in Figure 2E, S1C was included. (C) Genotypic analysis of embryos derived from E2f1+/-E2f7+/-_E2f8_-/- intercrosses at the indicated stages of development. (D) Genotypic analysis of embryos derived from p53+/-E2f7+/-_E2f8_-/- intercrosses at the indicated stages of development.

Figure 6

Figure 6

Microarray analysis of E10.5 embryos. (A) Heat maps of probe sets in Affymetrix microarrays that showed at least a 3-fold induction (left and middle) or a 3-fold reduction (right) of expression in _E2f7_-/-_E2f8_-/- relative to wild type embryos. Data are presented as the medium of 4 embryos from wild type, _E2f7_-/-E2f8+/+ and E2f7+/+_E2f8_-/- genotype groups, and the medium of 2 embryos from E2f7+/-E2f8+/- and _E2f7_-/-_E2f8_-/- genotype groups. Red and green indicate induction and reduction respectively. Probe sets representing genes of interest are indicated. (B) The expression of 8 target genes (6 up-regulated and 2 down-regulated) was evaluated by real-time RT-PCR assays. (C) Pie chart diagrams illustrate the non-random distribution of the stress-related genes among the total 88 up-regulated genes in DKO embryos.

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