canonical and atypical e2fs regulate the mammalian...

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ARTICLES Canonical and atypical E2Fs regulate the mammalian endocycle Hui-Zi Chen 1,2,10 , Madhu M. Ouseph 1,3,10 , Jing Li 1,11 , Thierry Pécot 1,4,5 , Veda Chokshi 1 , Lindsey Kent 1 , Sooin Bae 1 , Morgan Byrne 1 , Camille Duran 1 , Grant Comstock 1 , Prashant Trikha 1 , Markus Mair 1 , Shantibhusan Senapati 1 , Chelsea K. Martin 1 , Sagar Gandhi 1 , Nicholas Wilson 1 , Bin Liu 1,5 , Yi-Wen Huang 6 , John C. Thompson 1 , Sundaresan Raman 4 , Shantanu Singh 4 , Marcelo Leone 4 , Raghu Machiraju 4 , Kun Huang 5 , Xiaokui Mo 7 , Soledad Fernandez 7 , Ilona Kalaszczynska 8 , Debra J. Wolgemuth 9 , Piotr Sicinski 8 , Tim Huang 1 , Victor Jin 5 and Gustavo Leone 1,2,12 The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells. Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Using lineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles in vivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta and hepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These two antagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis and cytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cycles in mammals. The archetypal cell division cycle culminates in the generation of geneti- 1 cally identical diploid progenies. However, a number of cell-cycle varia- 2 tions in nature exist, with the endocycle being a remarkable variant used 3 by a wide range of organisms in diverse environmental contexts 1 . This 4 modified cell cycle is characterized by alternating DNA synthesis (S) and 5 gap (G) phases in the absence of intervening mitosis (M), karyokinesis 6 and cytokinesis. As a result of successive rounds of whole-genome 7 duplication, cells may achieve genome polyploidy that exceeds 1000C 8 (refs 2,3). The biological advantages of polyploidy have been proposed 9 to include an augmented response to strenuous cellular metabolic 10 demands; dampened sensitivity to apoptotic or checkpoint stimuli 4 ; 11 and buffering against deleterious mutations that may cause cancer 5,6 . Q1 12 The molecular mechanisms that control mitotic and variant cell 13 cycles among species are presumed to be similar. In the textbook view 14 1 Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA. 2 Medical Scientist Training Program and Integrated Biomedical Graduate Program, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA. 3 Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA. 4 Computer Science and Engineering, The Ohio State University, Columbus, Ohio 43210, USA. 5 Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA. 6 Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. 7 Center for Biostatistics, Office of Health Sciences, The Ohio State University, Columbus, Ohio 43210, USA. 8 Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02215, USA. 9 Department of Genetics and Development, Department of Obstetrics and Gynecology, The Institute of Human Nutrition, and The Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York 10032, USA. 10 These authors contributed equally to this work. 11 Present address: Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, California 94588, USA.. 12 Correspondence should be addressed to G.L. (e-mail: [email protected]) Received 13 September 2011; accepted 3 September 2012; published online XX Month XXXX; DOI: 10.1038/ncb2595 of cell-cycle control, E2F transcriptional activators and repressors are 15 portrayed to orchestrate a gene expression program essential for mitotic 16 cell-cycle progression 7 , with E2F activators and repressors oppositely 17 regulating a common set of target genes. However, careful analyses 18 of mouse knockout tissues demonstrate that E2F function in vivo 19 does not strictly adhere to this elegant dichotomous paradigm 8–16 . 20 This is perhaps not surprising given the large number of genes in the 21 mammalian E2F family, which consists of eight distinct genes encoding 22 at least nine structurally related protein products that can be divided 23 into activator or repressor subclasses 17 . Canonical E2F activators (E2F1, 24 E2F2, E2F3a and E2F3b) have a single DNA-binding domain (DBD), 25 heterodimerize with DP1/DP2 proteins and associate with co-activator 26 proteins to robustly induce RNA polymerase II-dependent gene 27 expression 18 . Canonical repressors (E2F4, E2F5 and E2F6) also have a 28 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1

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Page 1: Canonical and atypical E2Fs regulate the mammalian endocyclemedicine.osu.edu/bsgp/documents/Chen_Student_Highlights... · Canonical and atypical E2Fs regulate the mammalian ... hepatocytes

ART I C L E S

Canonical and atypical E2Fs regulate the mammalianendocycleHui-Zi Chen1,2,10, Madhu M. Ouseph1,3,10, Jing Li1,11, Thierry Pécot1,4,5, Veda Chokshi1, Lindsey Kent1, Sooin Bae1,Morgan Byrne1, Camille Duran1, Grant Comstock1, Prashant Trikha1, Markus Mair1, Shantibhusan Senapati1,Chelsea K. Martin1, Sagar Gandhi1, Nicholas Wilson1, Bin Liu1,5, Yi-Wen Huang6, John C. Thompson1,Sundaresan Raman4, Shantanu Singh4, Marcelo Leone4, Raghu Machiraju4, Kun Huang5, Xiaokui Mo7,Soledad Fernandez7, Ilona Kalaszczynska8, Debra J. Wolgemuth9, Piotr Sicinski8, Tim Huang1, Victor Jin5

and Gustavo Leone1,2,12

The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells.Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Usinglineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation(E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles invivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta andhepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These twoantagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis andcytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cyclesin mammals.

The archetypal cell division cycle culminates in the generation of geneti-1

cally identical diploid progenies. However, a number of cell-cycle varia-2

tions in nature exist, with the endocycle being a remarkable variant used3

by a wide range of organisms in diverse environmental contexts1. This4

modified cell cycle is characterized by alternatingDNA synthesis (S) and5

gap (G) phases in the absence of intervening mitosis (M), karyokinesis6

and cytokinesis. As a result of successive rounds of whole-genome7

duplication, cells may achieve genome polyploidy that exceeds 1000C8

(refs 2,3). The biological advantages of polyploidy have been proposed9

to include an augmented response to strenuous cellular metabolic10

demands; dampened sensitivity to apoptotic or checkpoint stimuli4;11

and buffering against deleteriousmutations thatmay cause cancer5,6.Q1 12

The molecular mechanisms that control mitotic and variant cell13

cycles among species are presumed to be similar. In the textbook view14

1Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University,Columbus, Ohio 43210, USA. 2Medical Scientist Training Program and Integrated Biomedical Graduate Program, College of Medicine, The Ohio State University,Columbus, Ohio 43210, USA. 3Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA. 4Computer Science and Engineering, TheOhio State University, Columbus, Ohio 43210, USA. 5Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA. 6Department of Obstetrics andGynecology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. 7Center for Biostatistics, Office of Health Sciences, The Ohio State University,Columbus, Ohio 43210, USA. 8Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston,Massachusetts 02215, USA. 9Department of Genetics and Development, Department of Obstetrics and Gynecology, The Institute of Human Nutrition, and The HerbertIrving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York 10032, USA. 10These authors contributed equally to this work.11Present address: Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, California 94588, USA..12Correspondence should be addressed to G.L. (e-mail: [email protected])

Received 13 September 2011; accepted 3 September 2012; published online XX Month XXXX; DOI: 10.1038/ncb2595

of cell-cycle control, E2F transcriptional activators and repressors are 15

portrayed to orchestrate a gene expression program essential formitotic 16

cell-cycle progression7, with E2F activators and repressors oppositely 17

regulating a common set of target genes. However, careful analyses 18

of mouse knockout tissues demonstrate that E2F function in vivo 19

does not strictly adhere to this elegant dichotomous paradigm8–16. 20

This is perhaps not surprising given the large number of genes in the 21

mammalian E2F family, which consists of eight distinct genes encoding 22

at least nine structurally related protein products that can be divided 23

into activator or repressor subclasses17. Canonical E2F activators (E2F1, 24

E2F2, E2F3a and E2F3b) have a single DNA-binding domain (DBD), 25

heterodimerize with DP1/DP2 proteins and associate with co-activator 26

proteins to robustly induce RNA polymerase II-dependent gene 27

expression18. Canonical repressors (E2F4, E2F5 and E2F6) also have a 28

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1

Leon05
Sticky Note
Q1; Please add the following affiliation correction for those annotated with superscript (1): Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Department of Molecular Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.
Page 2: Canonical and atypical E2Fs regulate the mammalian endocyclemedicine.osu.edu/bsgp/documents/Chen_Student_Highlights... · Canonical and atypical E2Fs regulate the mammalian ... hepatocytes

ART I C L E S

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Figure 1 Loss of E2f activators promotes TGC and hepatocyteendocycles. (a) NanoString analysis of TGC-specific E2f1–3 expression inlaser-capture-microdissected wild-type cells; n = 2 placentae analysedper time point. (b) Quantitative rtPCR analysis of E2f activatorsin pre- (E13.5) and postnatal (P0–12mo) wild-type livers, withn per time point as indicated. 3wk*, 1 day post-weaning (3wk).(c) Representative E9.5 H&E-stained placenta sections showing controland E2f1−/−;E2f2−/−;E2f3−/− (123tko) TGCs. The arrows point toselected TGCs. (d) Feulgen quantification of ploidy in E9.5 control and123tko TGCs; n = 3 per genetic group. One-way ANOVA, ∗P ≤ 0.05,∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. (e) Representative H&E-stained sections

showing control and Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f (Mx-123tko) liversof 8-week-old mice ten days after pIpC injection. (f) Measurementsshowing enlargedMx-123tko livers (top) and hepatocytes (bottom) relativeto control tissues and hepatocytes, with n per genetic group as indicated.Two-tailed Student t -test, ∗∗P ≤0.01. (g) Flow cytometry of control andMx-123tko liver nuclei from 8-week-old mice, with n per genetic group asindicated. One-way ANOVA, ∗∗P ≤0.01, ∗∗∗P ≤0.001. c control, wild-typeTGCs; e control, pIpC-injected Mx-cre;E2f1−/−;E2f2−/−;E2f3+/+ livers;f,g control, wild-type livers. Data in a,b,d,f,g are average values, ± s.d.are included where n>2 samples were analysed. Scale bars, 12.5 µm (c)and 10 µm (e).

single DBD, heterodimerize with DP1/DP2 proteins and associate with1

chromatin-modifying proteins to repress gene expression18. The most2

recently identified family members encode the atypical repressors E2F73

and E2F8, which have two tandem DBDs and repress gene expression4

independent of DP1/DP2 heterodimerization19.Q2 5

Whereas flies and plants use endocycles as a mechanism for cell6

growth in a variety of tissue types1,20, endocycles in mammals are7

mainly restricted to trophoblast giant cells (TGCs) in the placenta8

and hepatocytes in the liver1,5. Endocycles in these mammalian tissues9

are believed to be integral to organ physiology1,5; however, surprisingly10

little evidence exists to support this contention. Here we developed11

new genetic tools in mice and show by depletion of either canonicalQ3 12

E2F activators or atypical E2F repressors that these two separate E2F13

transcriptional programs converge to control the endocycle through14

the regulation of cellular events important for mitosis, karyokinesis and15

cytokinesis. Surprisingly, placentae and livers with severely restricted16

polyploidization retained sufficient physiological function to carry17

them through apparently normal development.18

RESULTS19

E2f1–3 ablation results in hyperploidy20

E2F-mediated transcription contributes to endocycle control in flies21

and plants1,5,19–21, but its role in mammals has not been thoroughly22

evaluated. Whole-genome duplication in mammalian endocycling 23

cells is temporally regulated during development with major ploidy 24

increases in TGCs during mid gestation and in hepatocytes following 25

weaning at around three weeks of age22,23. The levels of E2f1 and E2f2 26

messenger RNAs in laser-capture-microdissected TGCs fromwild-type 27

placentae, as measured by NanoString technology (Fig. 1a), were low 28

throughout placental development, whereas the level of E2f3mRNA 29

was high before embryonic day (E)8.5 and following E13.5. Expression 30

analysis in livers by quantitative real-time polymerase chain reaction 31

(rtPCR) showed relatively high levels of E2f1 and E2f2 during fetal 32

development and a precipitous decrease following birth (postnatal 33

day 0, P0), whereas expression of E2f3 (E2f3a and E2f3b) decreased 34

gradually following birth and was almost undetectable post-weaning 35

(Fig. 1b). Consistentwith the expression pattern of theirmRNAs, E2F3a 36

and E2F3b protein levels decreased in newborn pups post-weaning 37

(Supplementary Fig. S1a), and E2F1 and E2F2 proteins could not be Q4 38

detected beyond the intrauterine period (data not shown). Thus, it 39

seems that total E2F1–3 activator levels are minimized when TGCs and 40

hepatocytes are most actively endocycling. 41

We used targeted gene inactivation strategies in mice to rigorously 42

evaluate the role of E2F activators in endocycling TGCs and 43

hepatocytes. To avoid functional compensation24, we evaluated TGCs 44

in embryos deficient for the entire E2F activator subclass (E2f1–3). 45

2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

Leon05
Sticky Note
Q2. Fine as is (independent).
Leon05
Sticky Note
Q3; OK.
Leon05
Sticky Note
Q4, OK.
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ART I C L E S

Visual inspection of haematoxylin and eosin (H&E)-stained E9.51

E2f1−/−;E2f2−/−;E2f3−/− (123tko) placentae revealed the presence2

of TGCs with abnormally large nuclei (Fig. 1c). Feulgen staining3

and quantification of genome ploidy by standardized methods4

(Supplementary Fig. S1b) showed a further shift of the 123tko TGC5

population towards higher ploidy (Fig. 1d and Supplementary Fig. S1c).6

Analysis of 123tko TGCs at later stages of development was precluded7

owing to embryo lethality at E10.5 (refs 25,26).8

To evaluate the role of the three activator E2Fs in hepatocytes9

we used polyinosine-polycytidine (pIpC)-inducible Mx–cre mice2710

and conditional alleles of E2f3 (Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f;Mx-11

123tko) to examine the consequences of their combined ablation.12

Injection of pIpC in six-week-oldMx-123tkomice resulted in increased13

liver weights (Fig. 1f) and proportionately larger hepatocytes (Fig. 1e,f)14

than age-matched controls. Loss of E2f1 alone or in combination with15

E2f2 and E2f3 resulted in a gene-dose-dependent increase in hepatocyte16

ploidy (Fig. 1g and Supplementary Fig. S1d). The expression of E2f17

repressors (E2f4-8) was not affected by loss of E2f1–3 (Supplementary18

Fig. S1e). Together, these findings suggest that mammalian E2F19

activators have a physiologic role in suppressing endocycles.20

E2f7/E2f8 ablation reduces TGC ploidy21

Next, we explored the role of E2F repressors in endocycle control.22

Expression analysis of the canonical repressors (E2f4-6) showed23

relatively high levels of E2f4 in TGCs of E6.5 placentae that24

decreased with time, whereas E2f5-6 expression was relatively25

constant throughout placental development (Fig. 2a, left panel);26

expression of E2f4-6 was relatively high in fetal livers and their27

expression decreased after birth (Supplementary Fig. S2a). Inspection28

of E2f4−/− or E2f5−/− embryos and mice revealed normal placentae29

and livers (Supplementary Fig. S2b,c); E2f6−/− placentae and livers30

were not evaluated.31

We then focused our attention on the atypical repressors, E2F732

and E2F8. These E2Fs are expressed in TGCs throughout placental33

development (Fig. 2a, right panel). Immunohistochemistry showed34

robust staining of E2F7 and E2F8 proteins in a portion of TGCs (Fig. 2b,35

arrows), consistent with their established cell-cycle-dependent expres-36

sion in late S and G2 phases28–31. Visual inspection of E2f7−/−;E2f8−/−37

placentae at E10.5, one day before their embryonic lethality16, revealed38

a surprising number of TGCs at various stages of mitosis (Fig. 2c),39

implying an interruption of normal endocycles. Indeed, the ploidy40

of E2f7−/−;E2f8−/− TGCs never exceeded 64C (Fig. 2d, black bars),41

whereas wild-type TGCs with genomes >1,000C were readily detected42

(Fig. 2d, white bars). The combined inactivation of E2f7 and E2f843

led to ectopic bromodeoxyuridine (BrdU) incorporation (Fig. 2e,g),44

increased levels of G2/M (cyclin A2) and M-phase-specific proteins45

(cyclin B1 and phosphorylated H3, P-H3; Fig. 2f,g) and the appearance46

of numerous TGCs in metaphase and anaphase (Fig. 2h). Confocal47

microscopy and three-dimensional (3D) reconstruction of serially48

imaged E2f7−/−;E2f8−/− placentae showed that approximately 40%49

of TGCs contained at least two nuclei (Fig. 2i), which was confirmed50

by transmission electron microscopy (Fig. 2j). Placentae lacking E2f7,51

E2f8 or one allele of each (E2f7+/−;E2f8+/−) exhibited TGCs with52

an intermediate reduction in ploidy (Fig. 2d and Supplementary53

Fig. S2d,e) and a proportionate increase in G2/M- andM-phase-related54

events (Fig. 2g,j), once again suggesting a gene dosage effect.55

As the expression of trophoblast cell lineage markers was altered 56

in placentae globally deleted for E2f7 and E2f8 (TGCs, labyrinth 57

trophoblasts and spongiotrophoblasts)32, we could not formally rule 58

out a general disruption of differentiation as a possible cause for the 59

endocycle defects observed in mutant TGCs. To specifically ablate 60

E2f7 and E2f8 in TGCs of placentae we crossed Plfcre/+ knock-in 61

mice (Fig. 2k and data not shown)32 with E2f7f /f;E2f8f / f mice to 62

yield Plf-78dko embryos. We confirmed the TGC-specific ablation of 63

conditional alleles by PCR genotyping of laser-capture-microdissected 64

samples (Fig. 2l). The same abnormalities were observed in Plf- 65

78dko TGCs as in E2f7−/−;E2f8−/− TGCs, including the reduced 66

ploidy, increased cyclin A2, cyclin B1 and P-H3 expression levels, 67

and ectopic karyokinesis (Fig. 2d,g,j; red bars). In summary, these 68

data demonstrate cell autonomous functions for E2F7 and E2F8 in 69

facilitating TGC endocycles by repressing keymolecular events required 70

for mitosis and karyokinesis. 71

E2f7/E2f8 ablation prevents hepatocyte endocycles 72

The expression of E2f7 and E2f8 in fetal livers was high but decreased to 73

almost undetectable levels by weaning age (Fig. 3b and Supplementary 74

Fig. S3a). To examine their roles during postnatal liver development 75

we crossed E2f7f / f and E2f8f /f mice with Albumin–cre mice (Alb–cre; 76

ref. 33), which express cre in hepatocytes of late-term embryos and 77

newborn pups. The conditional inactivation of E2f7 (Alb-7ko), E2f8 78

(Alb-7ko) or both (Alb-78dko) did not significantly affect hepatic 79

mass (Supplementary Fig. S3b). However, evaluation of H&E- and 80

4,6-diamidino-2-phenylindole (DAPI)-stained liver sections from 81

Alb-8ko and Alb-78dko mice revealed markedly smaller hepatocytes 82

with smaller nuclei than in control animals (Fig. 3a,c). This reduction 83

in cell size was compensated by an increase in the total number of 84

hepatocytes (Fig. 3d). Moreover, Alb-8ko and Alb-78dko livers had 85

a decreased proportion of binucleated hepatocytes (Fig. 3e), which 86

is an early event believed to precede the onset of polyploidization34. 87

Strikingly, flow cytometry showed that hepatocytes in Alb-8ko and 88

Alb-78dko livers remained diploid over the entire lifetime of the 89

mouse, whereas hepatocytes in Alb-7ko livers had only a modest 90

reduction in genome ploidy (Fig. 3f). The percentages of Ki67-, 91

cyclin-A2-, cyclin-B1- and P-H3-positive hepatocytes were significantly 92

elevated in 2-month-old Alb-8komice and even more so in similarly 93

aged Alb-78dko mice (Fig. 3g). Analysis of mice globally deleted for 94

E2f7 (E2f7−/−) or E2f8 (E2f8−/−) yielded identical phenotypes as 95

conditionally deleted Alb-7ko and Alb-8komice (Supplementary Fig. 96

S3c,d). Surprisingly, Alb-78dkomice survived to old age in apparent 97

good health and retained the capacity to regenerate their livers on 98

chemical (carbon tetrachloride) or physical (partial hepatectomy) 99

injury (Supplementary Fig. S3e–h and data not shown). From these 100

results, we conclude that E2F8 is the principal atypical E2F that 101

functions to promote hepatocyte endocycles inmice. 102

The onset of genome endoreduplication in hepatocytes coincides 103

with the weaning of pups, but DNA content continues to increase 104

through adulthood even though very little E2f8 is expressed at this 105

time (see Fig. 3b). These observations raised the possibility that E2F8 106

might be required only for the programmed onset and not the 107

continuation of endocycles. We thus used a tamoxifen-inducible 108

system (SA-creERT2) to express cre in hepatocytes following birth 109

and/or the weaning of pups35. As determined by quantitative rtPCR, 110

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 3

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Page 4: Canonical and atypical E2Fs regulate the mammalian endocyclemedicine.osu.edu/bsgp/documents/Chen_Student_Highlights... · Canonical and atypical E2Fs regulate the mammalian ... hepatocytes

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–/–

+/+f/f

–/–

+/+f/f

20

40

0

50

30

10

Plfcre/+Control

Plfcre/+Control

×20

×20

×4

×4

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35

30

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5

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60

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20

10

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20

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P-H3/DAPI PL-1/DAPI

78dko Control

TGC: NanoString TGC: NanoStringa

d

e f h

i

g

j

l

k

b c

BrdU CycA2 CycB1 P-H3

×20

×40

E2f7+/+;E2f8+/+ E2f7–/–;E2f8+/+

Figure 2 E2f7 and E2f8 promote TGC endocycles. (a) NanoStringanalysis of TGC-specific E2f 4–6 (left panel) and E2f7/E2f8 (right panel)expression in laser-capture-microdissected wild-type cells; n=2 placentaeanalysed per time point. (b) Immunohistochemistry demonstrating E2F7(top left) and E2F8 (bottom left) expression in wild-type E10.5 TGCsbut not mutant controls (right panels). The arrows point to selectedTGCs. (c) Representative H&E-stained sections of E10.5 control andE2f7−/−;E2f8−/− (78dko) placentae. Inset, higher magnification ofthe outlined area, showing a 78dko TGC in metaphase. (d) Feulgenquantification of genome ploidy in E10.5 TGCs; n =3 placentae analysedper genetic group. (e–g) Immunostaining and quantification of S and Mphase proteins in E10.5 control and 78dko TGCs. The arrows point to TGCs.n = 3 placentae analysed per genetic group. (h) Co-immunofluorescencemicroscopy showing E10.5 78dko TGCs in anaphase (left, P-H3) andmetaphase (right, PL-1). DAPI stained total DNA. (i) Representativeconfocal images of nuclei in E10.5 control (top right) and 78dko TGCs

(top left) and 3D reconstruction of a binucleated 78dko TGC (bottom).Draq5 stained total DNA pseudocoloured in green. (j) Left, transmissionelectron micrograph (TEM) of a 78dko E10.5 TGC (left top, arrows indicatetwo nuclei; left bottom, enlarged view of the area outlined in the top imageshowing separation between nuclear envelopes). Right, quantification ofbinucleated E10.5 TGCs; n = 3 placentae analysed per genetic group.(k) X-gal staining of E10.5 control and Plfcre/+ placentae carrying thereporter allele Rosa26LoxP. (l) PCR genotyping of genomic DNA isolatedfrom laser-capture-microdissected E10.5 control and Plfcre/+ TGCs. e,f,icontrol, E2f7+/+;E2f8+/+; e,f,h–j 78dko, E2f7−/−

;E2f8−/−; k control,Plf+/+;Rosa26LoxP / + ; l control, Plf+/+;E2f7f / f;E2f8f / f; d,g,j Plf-78dko,Plfcre/+;E2f7f / f;E2f8f / f. PL-1, placental lactogen 1; N.E., nuclear envelope.Data in a,d,g,j are average values, ±s.d. are included where n>2 sampleswere analysed. One-way ANOVA, ∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001. Scalebars, 10 µm (b), 12.5 µm (e,f), 5 µm (h) and 10 µm (i). Uncropped imagesof blots are shown in Supplementary Fig. SXX.

supplying SA-creERT2;E2f8f / f mice with a tamoxifen diet beginning1

at either 7 days (2 weeks before weaning, pre-weaning) or 28 days2

of age (1 week following weaning, post-weaning) resulted in the3

tissue-specific efficient deletion of E2f8 (data not shown). Ablation of4

E2f8 pre-weaning prevented efficient polyploidization of hepatocytes5

(Supplementary Fig. S4a–c), whereas ablation post-weaning failed 6

to impact hepatocyte size, binucleation and DNA content (data not 7

shown and Supplementary Fig. S4a,b,d). These results suggest that 8

E2F8 is largely dispensable for the execution of endocycles following 9

the weaning of pups. 10

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ART I C L E S

12 m

o liv

er

H &

E

Un

its

3 p

er

nucle

us (

×1,0

00)

Num

ber

of

hep

. p

er

×40 f

ield

n = 3n = 3

n = 4

n =

61

n =

157

n = 3n = 4

n = 3

n = 2

n = 3

Rela

tive g

ene e

xp

ressio

n

Liver: quantitative rtPCR

Perc

en

tag

e o

f liv

er

nu

cle

i g

ate

d

Nu

mb

er

of

liver

nu

cle

i (×

1,0

00

)

Nu

mb

er

of

liver

nu

cle

i (×

1,0

00

)

3 wk n = 42 mo n = 912 mo n = 6 3 wk n = 4

2 mo n = 1012 mo n = 6

E13.5 P0

2 wk

3 w

k

3 wk∗

4 wk6

wk

12 m

o

E2f7E2f8

Alb-7ko Alb-8ko Alb-78dko

Perc

enta

ge o

f b

inucle

ate

d

hep

ato

cyte

s

Control

10

0

8

6

4

2

1.2 ∗∗∗

06 mo Confocal

0.8

Control

Alb-78dko0.4

400

300

200

100

02 mo 6 mo3 wk 12 mo

12 mo

n =

3n

= 3

n =

3

∗∗

∗∗∗∗∗∗ ∗∗∗

∗∗∗∗∗∗

∗∗∗

0

7

14

21

28

35

n =

3

n =

9n

= 2

n =

6n

= 7

n =

9

n =

5n

= 2

n =

6

n =

5

Alb-78dko

Perc

en

tag

e o

f

Ki6

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ositiv

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ep

.

Perc

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tag

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ositiv

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ep

.

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ositiv

e h

ep

.

∗∗∗

∗∗∗

∗∗

n =

19

0

0.5

0

1.0

n =

8n

= 7

n =

16

n =

8n

= 7

n =

7n

= 1

0n

= 6

n =

5n

= 8

n =

10

Control

Alb-78dko

E-cad/DAPI3 wk 2 mo 12 mo

n =

4 n =

8

n =

7n

= 6

3

2

1

0

1.0

0

0.5

0.8

0.4

1.2

Control

100

50

0

25

75

Perc

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tag

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f liv

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ate

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ate

d

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en

tag

e o

f liv

er

nu

cle

i g

ate

d

3 wk n = 42 mo n = 612 mo n = 5

4C 8C 16C

2C 4C 8C 16C2C

Control Alb-7koAlb-8ko Alb-78dko

12 mo 12 mo 12 mo

4C 8C16C2C 4C 8C 16C2C

2.0

0

2.5

1.5

1.0

0.5

Nu

mb

er

of

liver

nu

cle

i (×

1,0

00

)

Nu

mb

er

of

liver

nu

cle

i (×

1,0

00

)

0

1.5

1.0

0.5

2.0

0

1.5

1.0

0.5

2.0

2.5

1.5

1.0

0.5

0

a e

g

b

f

c d

Alb-78dAlb-8ko

Alb-7koControl

n =

6

n =

3n

= 3

n =

3n

= 3

n =

7n

= 2

n =

6n

= 8

n =

4n

= 3

n =

3n

= 5

n =

5n

= 5

n =

2

3 wk n = 42 mo n = 612 mo n = 2

100

50

0

25

75

4C 8C 16C2C 4C 8C2C 4C 8C 16C2C 4C 8C 16C2C16C

100

50

0

25

75

100

50

0

25

75

Figure 3 E2f8 is sufficient to promote hepatocyte endocycles.(a) Representative H&E-stained sections of aged (12 mo) livers.(b) Quantitative rtPCR analysis of E2f7 and E2f8 in pre- (E13.5)and postnatal (P0–12 mo) wild-type livers, with n per time pointanalysed as indicated. 3 wk*, 1 day post-weaning (3 wk). (c) Leftgraph, assessment of nuclei volume by confocal imaging and 3Dreconstruction in 6-month-old control and Alb-78dko hepatocytes. n,number of nuclei evaluated per genotype. Right panels, representativeconfocal images of DAPI-stained liver sections. (d) Assessment ofhepatocyte size in young (3 wk), adult (2–6 mo) and aged (12 mo)livers by quantifying the number of hepatocytes per image field,with n per genetic group analysed as indicated. (e) Left graph,quantification of binucleated hepatocytes in young, adult and aged

livers, with n per genetic group analysed as indicated. Right panels,immunofluoresence micrographs with anti-E-cadherin marking theboundary of mono- or binucleated hepatocytes. (f) Top row, flow cytometryof liver nuclei in young, adult and aged livers. n, number of liversanalysed per genotype and age group. Bottom row, representativefluorescence-activated cell sorting profiles of liver nuclei from aged (12mo) mice. (g) Immunohistochemistry and quantification of hepatocyteproliferation in 2-month-old livers, with n per genetic group analysed asindicated. Control, E2f7f / f;E2f8f / f; Alb-7ko, Alb–cre;E2f7f / f; Alb-8ko,Alb–cre;E2f8f / f; Alb-78dko, Alb–cre;E2f7f / f;E2f8f / f. Data in b–g areaverage values, ± s.d. are included where n >2 samples were analysed.One-way ANOVA, ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001. Scale bars,12.5 µm (a), 10 µm (c), 10 µm (e) and 10 µm (g).

E2F7/E2F8 regulate endocycle-related genes1

Given the role of E2F7 and E2F8 in transcriptional repression, we2

anticipated changes in gene expression as the principal underlying3

cause for the observed endocycle defects in E2f7/E2f8-deficient tissues.4

Gene expression profiling (Affymetrix Mouse Genome 430 2.0) using5

RNApurified from livers of 3-week-old wild-type,Alb-7ko,Alb-8ko and6

Alb-78dkomice showed that there were significantly more upregulated7

than downregulated genes in mutant tissues, consistent with their8

roles as repressors (Fig. 4a). Expression changes were confirmed9

by NanoString technology (Supplementary Fig. S5a). Supervised10

clustering methods identified three main classes of differentially 11

expressed genes (Fig. 4a). Class I includes genes differentially expressed 12

in all three mutant genetic groups relative to wild-type controls. These 13

genes may represent targets regulated by both E2F7 and E2F8 because 14

loss of eithermember led to their derepression. Class II represents genes 15

differentially expressed in two of the three mutant genetic groups and 16

includes target genes that may be uniquely repressed by either E2F7 17

or E2F8. Finally, class III represents genes differentially expressed in 18

only one mutant genetic group, and includes target genes that may be 19

synergistically repressed by E2F7 and E2F8. 20

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ART I C L E S

NanoString: E10.5 TGCs

Liv

er a

nd

TG

Cs (6

6)

G2

/M(3

3)

Genes a

nta

go

nis

tically re

gula

ted

P < 0.05

Genes antagonistically regulated

IPA: molecular and cellular functions

Number of genes

Affymetrix: liver

Cla

ss I

Cla

ss II

Cla

ss III

Syn

erg

.

targ

ets

E2F

8

targ

ets

E2F

7

targ

ets

E2F

7 a

nd

E2F

8 ta

rgets

Affymetrix: weaning age (3 wk) liver

1 2 3 4

P < 0.05

Cell cycle

Small molec. biochem.

Lipid metabolism

Nucleic acid metab.

Cell–cell signal.

1 2 3 1 2 3 4 1 2 3 4

Control 78dko 8ko 7ko

Control Alb-78dko Control Mx-123tko

10 20 30 40 50 600P < 0.00245

n = 2 n = 2 n = 2 n = 2

1 2 3 1 2 31 2 1 23 4

a b

c

d

Control Alb-78dko Alb-8ko Alb-7ko

P < 0.05–1.5 +1.5

–3.0 +3.0

–1.5 +1.5

Figure 4 Canonical activator and atypical repressor E2Fs regulate keytranscriptional networks coordinating endocycles. (a) Heat map ofapproximately 4,500 differentially expressed genes in weaning age(3 wk) livers. Class I: genes regulated by E2F7 and E2F8. Class II:genes regulated by either E2F7 or E2F8. Class III: genes synergisticallyregulated by E2F7/E2F8. n = 3–4 livers analysed per genetic group.Control, E2f7f / f;E2f8f / f; Alb-78dko, Alb–cre;E2f7f / f;E2f8f/ f; Alb-8ko,Alb–cre;E2f8f / f; Alb-7ko, Alb–cre;E2f7f / f. (b) Top, heat map of TGCgene expression in a custom NanoString mRNA code set. RNA wasisolated from laser-capture-microdissected E10.5 TGCs in frozen

placental tissues. Bottom, G2/M-related genes in NanoString codeset with deregulated expression in TGCs. n = 2 placentae analysed pergenetic group. Control, E2f7+/+

;E2f8+/+;78dko, E2f7−/−;E2f8−/−; 8ko,E2f7+/+;E2f8−/−; 7ko, E2f7−/−;E2f8+/+. (c) Heat map of downregulatedgenes in Mx-123tko livers with significantly deregulated expression inAlb-78dko livers. n =3–4 livers analysed per genetic group. Alb-78dko,Alb–cre;E2f7f /f;E2f8f/f; Mx-123tko, Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f.(d) Top five molecular and cellular functions revealed through IngenuityPathway Analysis (IPA) of genes antagonistically regulated by E2F1–3and E2F7/E2F8.

Further analysis focused on genes upregulated in both Alb-8ko1

and Alb-78dko livers (Class II), as hepatocytes in these two genetic2

groups exhibited the most pronounced reduction in genome ploidy,3

as well as genes upregulated in all three mutant cohorts (in Class4

I), because these could also contribute to the observed defect in5

polyploidization. Closer inspection of these genes by Ingenuity Pathway6

Analysis revealed a striking bias for functions related to cytokinesis,7

G2/M progression and various stages of mitosis, such as chromosome 8

condensation, stabilization and segregation (Supplementary Fig. S5b,c). 9

By manual annotation we also noted an increase in gene products with 10

GTPase-activating (GAP) or GTP exchange factor (GEF) functions 11

(data not shown), which regulate the Rho family of small GTPases 12

essential for successful cytokinesis36. NanoString technology was 13

then used to interrogate the expression of a panel of genes with 14

6 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

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ART I C L E S

Rela

tive o

ccup

ancy

Con. F7 F8 Con. F7 F8 Con. F7 F8 Con. F7 F8

ChIP E2F targets in HepG2 cells

Rcho-1 TS

Wild-type liver

HepG2

Rela

tive o

ccup

ancy

ChIP E2F targets in vivo

24 h post

TV inject.:

Flag

Tub.

IB:

Cdk1 Ccna2 TubCcne2 Chek1

ChIP E2F targets in Rcho-1 TS cells

Flag

Tub.

IB:

IB:

Flag

Tub.

Transf: Con. F7 F8

Con. F7 F8

Con. F8

Transf:

24

18

12

6

0

Rela

tive o

ccup

ancy

Con. F7 F8

Con. F7 F8 Con. F7 F8

16

12

6

0

14

7

0

12

8

4

0

12

8

4

0

E2f1 Ccna2 TubCcne2 Chek1

24

18

12

6

0

24

18

12

6

0

20

15

10

5

0

45

30

15

0 0

10

20

30

40

Con. F7 F8 Con. F7 F8 Con. F7 F8

10

5

0

5

3

0

4

2

1

3

0

2

1

Ccne2 Cdk1E2f1

a b

c d

e f

Con. F8 Con. F8 Con. F8

IgG

Anti-Flag

IgG

Anti-Flag

IgG

Anti-Flag

Figure 5 Atypical repressors E2F7/E2F8 directly bind gene targetsinvolved in endocycle control. (a) Immunoblot of transfected HepG2cells showing exogenous expression of Flag-tagged E2F7 and E2F8proteins with tubulin as the control. The arrows indicate tagged protein.(b) Representative ChIP assay in transfected HepG2 cells with anti-Flagantibodies demonstrating enhanced occupancy of Flag-tagged E2F7 andE2F8 proteins on E2F-binding sites in the promoter of G1/S (E2f1and Ccne2 ) and G2/M (Ccna2 and Chek1) genes. The tubulin (Tub)gene, a non-E2F target, demonstrates specific recruitment of Flag-taggedE2F7 and E2F8 to target promoters containing consensus E2F-bindingsequences. n = 3 independent transfection–ChIP experiments performed.(c) Immunoblot of transfected Rcho-1 trophoblast stem (TS) cells showingexogenous expression of Flag-tagged E2F7 and E2F8 proteins with tubulinas the control. The arrows indicate tagged protein. (d) RepresentativeChIP assays in transfected Rcho-1 trophoblast stem cells of G1/S and

G2/M genes as in HepG2 cells. n = 3 independent transfection–ChIPexperiments performed. (e) Immunoblot of liver lysates from 6-week-oldmice demonstrating expression of tagged E2F8 protein. A total of 10 µgof plasmid DNA was delivered through tail vein (TV) injection. Lysateswere prepared from livers 24h after injection. Tubulin served as a loadingcontrol. n = 3 and n = 5 wild-type mice were injected with control(empty) plasmid and plasmid expressing Flag–E2F8, respectively. Oneout of 5 had detectable expression of Flag-tagged E2F8 as shown bywestern blotting. (f) ChIP assay in liver lysates with anti-Flag antibodiesdemonstrating enhanced occupancy of Flag-tagged E2F8 on E2F-bindingsites in the promoter of G1/S (E2f1 and Ccne2 ) and G2/M (Cdk1) genes.a–f Con., empty plasmid; F7, plasmid expressing Flag-tagged E2F7; F8,plasmid expressing Flag-tagged E2F8. Data in b,d,f from the representativeexperiment are average values from triplicate quantitative rtPCR reactions±s.d. Uncropped images of blots are shown in Supplementary Fig. SXX.

cell-cycle- and G2/M-related functions in TGCs from E10.5 wild-type,1

E2f7−/−, E2f8−/− and E2f7−/−;E2f8−/− placentae. As shown in Fig. 4b,2

66 of the 99 genes interrogated had predominantly increased expression3

in E2f7/E2f8-deficient TGCs, suggesting a commonmechanism for how4

E2F7 and E2F8may regulate endocycles in TGCs and hepatocytes.5

A significant portion of common upregulated genes in E2f7/E2f8-6

deficient TGCs and hepatocytes have E2F-binding elements on their7

promoters, raising the possibility that these may represent direct8

targets of E2F7/E2F8. To test this hypothesis, we transfected human9

HepG2 cells and rat Rcho-1 trophoblast stem cells with plasmids10

expressing Flag-tagged E2F7 or E2F8 and performed chromatin11

immunoprecipitation (ChIP) assays with anti-Flag antibodies. As12

shown in Fig. 5b,d, Flag-specific antibodies, but not control IgG,13

co-immunoprecipitated promoter sequences containing E2F-binding 14

elements, but not irrelevant sequences lacking E2F-binding sites (Tub). 15

The above results were corroborated by further ChIP assays in human 16

embryonic kidney (HEK) 293 cells transfected with either Flag-tagged 17

E2F7 or E2F7 (Supplementary Fig. S5d) and in wild-type liver tissues 18

transfected by hydrodynamic-based methods with plasmids expressing 19

Flag-tagged E2F8 (Fig. 5e,f). Together, these results suggest that E2F7 20

and E2F8 repress a core group of genes with prominent involvement in 21

the regulation of G2/M-related events. 22

E2F1 functionally opposes atypical E2F7/E2F8 23

Given that ablation of E2f1–3 and E2f7/E2f8 led to opposite effects 24

on hepatocyte ploidy, we explored the possibility of cross-regulation 25

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ART I C L E S

1ko Alb-178tkoAlb-78dkoControl

6 m

o

H &

E

Num

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ld

Perc

en

tag

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f h

ep

atic m

ass

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enta

ge o

f liv

er

nucle

i g

ate

d

Control n = 4

Alb-178tko n = 3

1ko n = 5Alb-78dko n = 5

∗∗

∗∗ ∗ ∗∗∗

∗∗∗∗∗

∗∗∗

∗∗∗

Perc

en

tag

e o

f T

GC

s w

ith

estim

ate

d p

loid

y

Perc

enta

ge o

f p

ositiv

e

TG

Cs ∗∗∗

E1

0.5

H

& E

Plf-178tko

Perc

en

tag

e o

f p

ositiv

e

hep

ato

cyte

s

Perc

en

tag

e o

f

bin

ucela

ted

hep

.

Control Plf-78dko1ko

20

40

0

60Control n = 3

1ko n = 3Plf-78dko n = 3

Plf-178tko n = 3

45

0

36

27

18

9

35

0

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14

7

25

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20

15

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20

BrdU CycA2P-H3Karyok.

7

0

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4

3

2

1

400

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100

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50

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25

4C 8C 16C2C 32C

45

0

36

27

18

9

3.0

0

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1.4

0

0.7

1.2

0

0.6

1.0

0

0.5

2-4C 8C 16C 32C 64C 128C 256C 512C 1,024C

Ki67 P-H3 CycB1CycA2

a

c

d

b

e

g h

f

Figure 6 Loss of E2f1 restores endocycles in E2f7/E2f8 deficient TGCsand hepatocytes. (a) Representative H&E-stained sections of E10.5 TGCs.The lower left insets show higher magnification views of the areas outlinedin the main panels. (b) Feulgen quantification of genome ploidy inE10.5 TGCs; n = 3 placentae per genetic group analysed. Note thesignificant increase in the proportions of 64C and 128C Plf-178tko TGCsrelative to Plf-78dko TGCs. (c) Quantification of E10.5 TGCs undergoingkaryokinesis and expressing S/M phase markers; n = 3 placentae pergenetic group analysed. Note the significant reduction in the proportionof Plf-178tko TGCs undergoing karyokinesis relative to Plf-78dko TGCs.(d) Representative H&E-stained liver sections from 6-month-old mice.(e) Measurements showing no significant change in the percentageof hepatic mass in 6-month-old mice and a significant decrease inthe number of hepatocytes per image field in Alb-178tko relative to

Alb-78dko livers, with n per genetic group analysed as indicated inf. (f) Flow cytometry of liver nuclei in 6-month-old mice, with n pergenetic group analysed as indicated in the legend. Note the significantincrease and decrease in the proportion of 4C and 2C nuclei, respectively,in Alb-178tko livers relative to Alb-78dko livers. (g) Quantification ofbinucleated hepatocytes in 2-month-old livers, with n per genetic groupanalysed as indicated in f. (h) Quantification of hepatocyte proliferationin 2-month-old livers, with n per genetic group analysed as indicatedin Fig. 6f. a–c Control, E2f7+/+;E2f8+/+; d–h Control, E2f7f / f;E2f8f / f;1ko, E2f1−/−; Plf-178tko, Plfcre/+;E2f1−/−;E2f7f /f;E2f8f / f; Alb-178tko,Alb–cre;E2f1−/−;E2f7f / f;E2f8f / f. Data in b,c,e–h reported as average± s.d.b,f one-way ANOVA, ∗P ≤0.05;∗∗P ≤0.01; ∗∗∗P ≤0.001. c,e,g,h two-tailedStudent t -test, ∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001. Scale bars, 12.5 µm(a) and 10 µm (d).

between these two E2F subclasses. Loss of E2f1–3 did not impact1

the expression of other E2Fs (E2f4-8; see Supplementary Fig. S1e);2

however, loss of E2f7/E2f8 resulted in a transient increase of E2f13

expression soon after birth in the liver (Supplementary Fig. S5e).4

Interestingly, loss of E2f7/E2f8 had no significant effect on the5

expression of E2f1–6 in TGCs (Supplementary Fig. S5f). We then6

profiled global gene expression in hepatocytes lacking the three E2F7

activators (Mx-123tko). Strikingly, this analysis showed that most genes8

upregulated in E2f7/E2f8-deficient hepatocytes were downregulated9

in E2f1–3-deficient hepatocytes (Fig. 4c), and many of these genes10

had annotated cell-cycle functions related to G2/M transitions or11

mitoses and had E2F-binding sites in their promoters (Fig. 4d and12

Supplementary Fig. S5g,h).13

On the basis of the above results we considered the hypothesis14

that canonical E2F activators may functionally antagonize atypical15

E2F repressors in the control of endocycles. To rigorously test this16

possibility we generated and analysed mice with TGCs or hepatocytes17

deficient for E2F7, E2F8 and E2F1 (E2f1−/−). The ablation of E2f7f / f 18

and E2f8f / f alleles in TGCs was achieved by Plfcre/+ (Plf-178tko) and 19

their ablation in hepatocytes was achieved by Alb–cre (Alb-178tko). 20

Placenta sections from E10.5 Plf-178tko embryos exhibited TGCs 21

with large nuclei having increased ploidy (Fig. 6a), with some TGCs 22

having 128C and 256C genomes (Fig. 6b), which was never observed 23

in Plf-78dko TGCs. This increase in ploidy was accompanied by 24

a significant decrease in the number of multinucleated TGCs and 25

a slight decrease in BrdU- and P-H3-positive cells, even though 26

the number of cyclin-A2-positive TGCs was not reduced (Fig. 6c). 27

Similar results were obtained when placentae globally deleted for 28

E2f1, E2f7 and E2f8 (178tko) were examined (Supplementary Fig. 29

S6a–c). Evaluation of Alb-178tko livers revealed larger hepatocytes 30

containing greater DNA content than in Alb-78dko livers, with 31

some reaching 8C ploidy (Fig. 6d–f). The Alb-178tko livers also 32

had many more binucleated hepatocytes (Fig. 6g), and importantly, 33

had fewer Ki67-, P-H3- and cyclin-B1-positive hepatocytes, even 34

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ART I C L E S

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2–4C 8C 16C 32C 64C 128C 256C 512C 1,024C

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a b

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d

Figure 7 Cyclin A ablation reinstates genome ploidy of E2f7/E2f8 -deficientTGCs and hepatocytes. (a) Images of H&E-stained E10.5 TGCs. Thecombined loss of Ccna1 and Ccna2 (Plf-A12dko) led to TGCs similarin appearance to control TGCs. The further loss of Ccna1 and Ccna2in E2f7/E2f8 -deficient TGCs (Plf-A1278qko) led to TGCs with anormal (wild-type-like) appearance, in contrast to Plf-78dko TGCsthat seemed small or were binucleated (yellow arrows). The lower leftinsets show higher magnifications of the areas outlined in the mainpanels. (b) Feulgen quantification of genome ploidy in E10.5 TGCs. Theintensities (that is, estimated genome content) of 120–140 TGCs werequantified per placenta sample, with n placenta samples analysed pergenetic group as indicated. A number of TGCs quadruply deficient forCcna1, Ccna2, E2f7 and E2f8 reach ploidy levels of 128C and 256C.Control, E2f7f / f;E2f8f / f; Plf-A12, Plfcre/+;Ccna1−/−;Ccna2f / f; Plf-78dko,Plfcre/+;E2f7f / f;E2f8f / f;Plf-A1278qko, Plfcre/+;Ccna1−/−;Ccna2f / f;E2f7f / f;E2f8f / f. (c) Images of H&E-stained 3-month-old liver sections, which

were stained with the Feulgen technique to facilitate quantification of DNAcontent, showing an increase in both cellular and nuclear size of hepatocyteslacking Ccna1 and Ccna2 (Alb-A12dko) when compared with controlhepatocytes. Hepatocytes quadruply deficient for Ccna1, Ccna2, E2f7 andE2f8 (Alb-A1278qko) had nuclei resembling control hepatocytes. (d) Feulgenquantification of genome ploidy in livers of 3-month-old mice. The intensities(that is, estimated genome content) of 100 hepatocyte nuclei were quantifiedper liver sample, with n liver samples analysed per genetic group as indicated.Ccna1/Ccna2 -deficient livers had an increased proportion of hepatocyteswith higher ploidy levels (16C, 32C and 64C) relative to control livers.Quadruple deficient livers had a significantly elevated number of hepatocyteswith 16C genomes relative to Alb-78dko livers. Control, E2f7f /f;E2f8f / f;Alb-A12,Alb–cre;Ccna1−/−;Ccna2f / f; Alb-78dko, Alb–cre;E2f7f /f;E2f8f / f;Alb-A1278qko, Alb–cre;Ccna1−/−;Ccna2f / f;E2f7f / f;E2f8f / f. Data in b,dare average values, ±s.d. are included where n >2 samples were analysed.One-way ANOVA, ∗P ≤0.05; ∗∗∗P ≤0.001. Scale bars, 12.5 µm.

though again, the percentage of cyclin-A2-positive cells was not1

reduced (Fig. 6h). Thus, it seems that loss of E2f1 suppressed some,2

but not all, mitotic defects in TGCs and hepatocytes caused by3

E2f7/E2f8 deficiency.4

Loss of cyclin A2 reinstates a G2/M block and polyploidy in5

E2f7/E2f8 -deficient cells6

As the inactivation of E2f7/E2f8 results in ectopic mitoses without7

inhibiting DNA replication, we predicted that re-establishing a8

mitotic block would restore polyploidy in E2f7/E2f8-deficient cells.9

Previous analysis of trophoblast stem cell cultures showed that p57KIP210

activity participates in driving TGC differentiation and endocycles11

by inhibiting Cdk1 function37. However, we failed to detect any12

measurable change in p57KIP2 mRNA or protein levels that might13

underlie the phenotype of E2f7/E2f8-deficient TGCs or hepatocytes14

(Supplementary Fig. S6d). Given that cyclin A activity is critical15

for G2/M progression by regulating the APC (also known as theQ5 16

cyclosome)38, we sought to evaluate the consequence of inactivating17

cyclin A1/A2 in E2f7/E2f8-deficient TGCs and hepatocytes. We thus18

intercrossed Ccna1−/−;Ccna2f / f mice39 with Plfcre/+;E2f7f /f;E2f8f / f and19

generated embryos with quadruply deficient TGCs (Plf-A1278qko).20

Remarkably, TGCs in E10.5 Plf-A1278qko embryos had larger nuclei21

with greater DNA content than TGCs in Plf-78dko embryos, with some22

reaching 1024C (Fig. 7a,b). We also intercrossed Ccna1−/−;Ccna2f / f 23

mice with Alb–cre;E2f7f / f;E2f8f / f mice to generate offspring with 24

quadruply deficient hepatocytes (Alb-A1278qko). As shown in Fig. 7d, 25

hepatocytes in 3-month-old Alb-A1278qko mice had similar ploidy 26

levels as in age-matched wild-type mice. Interestingly, the loss of 27

Ccna1/Ccna2 alone (Alb-A12dko) in hepatocytes resulted in hyperploidy 28

(Fig. 7c,d). In summary, inhibiting the transcriptional network that 29

signals G2/M progression (by E2f1 inactivation) or interfering with 30

the mitotic machinery (by Ccna1/Ccna2 inactivation) re-established 31

a mitotic block and reinstated higher levels of polyploidy in 32

E2f7/E2f8-deficient cells. 33

DISCUSSION 34

In multi-cellular organisms there exists diversity in the kinetics and 35

composition of cell cycles and, presumably, in the mechanisms that 36

regulate them. The endocycle is commonly used in flies and plants and, 37

hence, more is known about how they are controlled in these organisms 38

than in mammals40–45. The balanced action of the E2F activator (dE2f1) 39

and repressor (dE2f2) in Drosophila is critical for the tight regulation of 40

cyclin E expression and successful completion of S phase in mitotic and 41

endocycling cells21,42,43. Although E2Fs are also involved in controlling 42

endocycles in plants, their role seems to be different from in mammals. 43

For example, the atypical E2Fe repressor in Arabidopsis is required to 44

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ART I C L E S

prevent endocycles44, whereas we show that the atypical E2F7 and E2F81

family members in mice promote endocycles. A comparison of the2

regulation of how atypical E2Fs are expressed during development3

and identification of their direct targets might shed light on these4

differences. In summary, our present work exposes an intricate E2F5

network involving balanced and antagonistic activities of canonical E2F6

activators (E2F1–3) and atypical E2F repressors (E2F7-8) that controls7

mammalian endocycles. Perhaps the most pronounced manifestation8

of altering the balance in the E2F network is the ectopic mitosis and9

karyokinesis observed in E2f7/E2f8-deficient TGCs.10

The results presented here expose two distinct arms of the E2F11

network, one arm involving canonical activators (E2F1–3) and a12

second involving atypical repressors (E2F7/E2F8), that converge to13

antagonistically regulate a common gene expression program critical14

for mammalian endocycles in vivo. A similar antagonistic role for E2F15

activators and atypical repressors seems to be at play in the control of a16

distinct program that is critical for mitotic cell cycles during placental17

development32. As loss of E2f4 or E2f5 in mice12–14 did not seem toQ6 18

affect the polyploidization of TGCs and hepatocytes (Supplementary19

Fig. S2c and data not shown) we propose that the most ancient20

E2F repressor arm consisting of atypical E2f7 and E2f8 evolved to21

specifically regulate variant cell cycles inmetazoans in amanner distinct22

from the function of canonical E2F repressor proteins. One reason23

for this might be to keep E2F7/E2F8-mediated repression outside24

the influence of the cyclin–CDK axis. As E2F7/E2F8 DNA-binding25

activity is independent of DP proteins, atypical repressors would26

remain unresponsive to CDK-mediated phosphorylation of DP, which27

negatively regulates the DNA binding and functions of canonical28

repressors45–47, allowing repression at a time (S/G2) when CDK29

activity is high. However, the exact molecular mechanism of how30

canonical activators and atypical repressors coordinate gene expression31

in endocycling tissues remains obscure.32

It has been assumed that polyploidy in mammals, as in flies and33

plants, is integral to the physiologic functions of placentae and livers.34

Here we generated mouse models with significantly reduced TGC35

and hepatocyte ploidy to assess this hypothesis. Surprisingly, TGCs36

and hepatocytes having significantly restricted ploidy levels seemed to37

impart placentae and livers, respectively, with adequate organ function38

to carry animals through embryonic and adult development. Whether39

altered liver ploidy in mice impacts ageing-related processes such as40

tumour development remains to be evaluated. In summary, genetic,41

systems-based and biochemical approaches revealed a fundamental42

role for the canonical activator and atypical repressor arms of the E2F43

network in orchestrating mammalian endocycles in vivo. �44

METHODS45

Methods and any associated references are available in the online46

version of the paper.47

Note: Supplementary Information is available in the online version of the paper48

ACKNOWLEDGEMENTS49

We thank L. Rawahneh, J. Moffitt and N. Lovett for excellent technical assistance50

with histology, and P. Wenzel for generating and collecting 123tko placentae.51

We also thank these individuals from OSUCCC Shared Resources: T. Wise, J.52

Palatini, H. Alders, P. Yan, P. Fada and B. Rodriguez (Microarray and Nucleic Acid53

Shared Resources); B. McElwain (Analytic Cytometry); R. Burry, K. Wolken, and B.54

Kemmenoe (Microscopy and Imaging); and K. La Perle (Comparative Pathology

and Mouse Phenotyping). We are grateful for PL-1 antibodies provided by F. 55

Talamantes, Rcho-1 trophoblast stem cells from M. Soares and HepG2 cells from Q7 56

S. Jacobs. This work was funded by NIH grants to G.L. (R01CA85619, R01CA82259, 57

R01HD047470, P01CA097189) and P.S. (R01CA132740). G.L. is a recipient of 58

The Pew Charitable Trust Scholar Award and the Leukemia & Lymphoma Society 59

Scholar Award. H-Z.C., M.M., S.S., S.R. and T. P. are recipients of the Pelotonia 60

Fellowship Award. 61

AUTHOR CONTRIBUTIONS 62

H-Z.C., M.M.O., J.L. and G.L. designed the experiments. H-Z.C., M.M.O., J.L., 63

T.P., V.C., L.K. and S.B. performed experiments. H-Z.C. and M.M.O. co-wrote the 64

paper with G.L.; all other authors listed helped perform experiments. Specifically, 65

J.C.T., S.R., S.S., M.L., R.M. and K.H. helped with experiments relating to 66

confocal microscopy and 3D reconstruction. X.M. and S.F. helped analyse data and 67

performed statistics. I.K., D.J.W. and P.S. provided mice for the study and reviewed 68

drafts of the manuscript. T.H., B.L. and V.J. contributed to discussion relating to 69

Affymetrix/NanoString analyses. 70

COMPETING FINANCIAL INTERESTS 71

The authors declare no competing financial interests. Q8 72

Published online at www.nature.com/doifinder/10.1038/ncb2595 73

Reprints and permissions information is available online at www.nature.com/reprints 74

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METHODS DOI: 10.1038/ncb2595

METHODS1

Mouse husbandry and genotyping. All mice were housed in barrier conditions2

with a 12-h light/dark cycle and had access to food/water ad libitum. Genotyping3

primers are provided in Supplementary Table S1.4

Feulgen analysis. Placenta/liver sections of 5 µm were treated with 1N hy-5

drochloric acid and stained with Schiff reagent. Cytoplasm was counterstained6

with aqueous light green (1%). Nuclear intensity was quantified using ImageJ7

(http://rsbweb.nih.gov/ij/; ref. 48). TGC ploidy was expressed relative to 2C–4C8

labrynthine trophoblast cells. For binucleated TGCs, ploidy levels of individual9

nuclei were summed to derive cumulative ploidy. Hepatocyte ploidy was expressed10

relative to 2C–4C hepatocytes deficient for E2f7/EfF8. Hepatocyte ploidy was11

expressed on a per nucleus basis.12

Flow cytometry analysis. Nuclei suspensions were obtained from frozen liver13

tissue as previously described49. The total DNA content of a minimum of 30,00014

nuclei per liver samplewas analysed using LSR II (BDBiosciences). Cell cycle profiles15

were generated using FlowJo (Tree Star).16

Affymetrix analyses. Total RNA was extracted from a minimum of 3 livers per17

genetic group using TRIzol (Invitrogen). The RNA quality was verified using an18

Agilent 2100 Bioanalyzer, and samples were submitted to the OSUCCCMicroarray19

Shared Resource facility for biotinylated complementary RNA generation and20

MOE4302.0 hybridization. AGeneChip Scanner 3000 (Affymetrix)was used for data21

acquisition. Pre-processing of raw signal intensities was performed using the robust22

multi-chip average method50. BRB-Array Tools 3.8.1 was then used to identify23

differentially expressed genes51. For heat map images, the geometric mean of genes24

after robust multi-chip average normalization from control liver samples was set25

as a reference, with colours corresponding to the ratio between expression of genes26

in mutant samples and the reference. Functional annotation was performed using27

Ingenuity Pathway Analysis (Ingenuity Systems).28

NanoString analyses. From each tissue sample, total RNA (200 ng) was profiled29

on a custom code set containing 200 selected genes following the manufacturer’s30

instructions (Supplementary Table S3). For TGC gene expression analysis, we31

isolated TGCs from frozen placenta sections using laser capture microdissection32

(PALM MicroLaser system). Total RNA was then extracted using the Arcturus33

PicoPure RNA Isolation Kit (Applied Biosystems 12204-01). For validation of34

gene expression changes in the liver (Supplementary Fig. S4a), the same set of35

TRIzol-isolated RNA was used in both Affymetrix and NanoString assays. The36

NanoString data are first normalized to the geometric mean of positive control spike37

counts in each lane of the cartridge, followed by subtracting the minimum negative38

control spike counts in each lane to remove the background. Finally, expression39

of each gene in the code set is normalized with respect to the geometric mean of40

the housekeeping genes. Heat maps were generated in the same manner as for the41

Affymetrix gene expression data.42

Confocal microscopy and 3D reconstruction. Tissues sections of 70 µm were43

stained with 1mM Draq5 (Biostatus) at 4 ◦C overnight. Sections were examined44

by confocal laser scanning microscopy (Zeiss LSM 510) at 63× and 0.7× scanQ9 45

zoom. Optical sections with between-plane-plane resolution of 0.40 µm and axial46

resolution of 0.42 µm were acquired. Four to five non-overlapping regions per47

placenta were imaged with a field view of 207 µm×207 µmand a depth of 40±2 µm,48

resulting in a stack of 100 images for each region. This process was identically49

repeated for liver samples. For 3D reconstruction, the image stacks were processed50

using Insight Toolkit and ITK-SNAP (ref. 52). Each nucleus was segmented by active51

contour segmentation using region competition as the stopping criterion53. Nuclei52

of binucleated TGCs were constructed separately and assigned different colours.53

Only the volumes of individual hepatocyte nuclei were measured.54

Transmission electron microscopy. Placentae were fixed/processed following a55

standard protocol (http://cmif.osu.edu/419.cfm) and embedded in Eponate resin.56

Placental cross-sections of 70 nm were mounted on copper grids, double stained57

with uranyl acetate and lead citrate, and observed with a Technai G2 Spirit58

transmission electron microscope (FEI).59

Transient transfections. For HepG2, subconfluent cells were transfected with60

1:3 ratio of plasmid DNA/reagent (X-tremeGENE HP DNA Transfection Reagent61

(Roche)) in 10% FBS RPMI-1640. Cells were collected 48 h after transfection. In62

one experiment, three 100 cm plates of transfected cells were combined for lysis and63

sonication as one sample in the ChIP assay. For Rcho-1, transfection conditions64

were the same as described for HepG2 cells. Rcho-1 cells were maintained as65

previously described54. In one experiment, two 150 cm plates were combined as66

one sample used in the ChIP. A total of three independent experiments for each67

cell line was performed. For HEK293 cells, subconfluent cells in 10% FBS DMEM

were transfected using calcium phosphate with 25 µg of plasmid DNA expressing 68

wild-type Flag–E2F7/Flag–E2F8 or mutant Flag–E2F7/Flag–E2F8. 69

Systemic administration of plasmid DNA. Six-week-old wild-type male mice 70

were injected with either sterile PBS or sterile PBS containing 10 µg plasmid DNA 71

through the tail vein following a published protocol55. Mice were euthanized by CO2 72

asphyxiation 24 h after tail-vein injection. Whole-body perfusion with warm saline 73

was performedusing the intracardiacmethod before excision of the liver, subsequent 74

derivation of a single-celled suspension by passing the perfused tissue through a 75

40 µmmesh and fixation/processing for ChIP assays. 76

ChIP assays. For HepG2 and Rcho-1 trophoblast stem cell lines, transfected 77

cells were crosslinked in growth media containing 1% formaldehyde followed 78

by neutralization with 1.25M glycine. The cytoplasmic fraction was discarded 79

(cytoplasmic lysis buffer: 5mM PIPES pH 8.0, 85mM KCl, 0.5% NP40 and fresh 80

protease inhibitors). Nuclear lysates were collected by incubating the nuclei pellet in 81

lysis buffer containing 50mM Tris at pH 8.0, 10mM EDTA, 0.3% SDS and a fresh 82

cocktail of protease/phosphatase inhibitors. Sonicated chromatin (∼100–300 base 83

pairs (bp)) was incubated with 10 µg of anti-Flag mouse monoclonal antibodies 84

(Sigma F1804) or normal mouse IgG (Santa Cruz sc-2025) at 4 ◦C overnight. 85

Antibody–protein–DNA complexes were recovered by the addition of 50% protein 86

G agarose slurry (Millipore 16-266) pre-cleared with 10mgml−1 transfer RNA and 87

10mgml−1 BSA. De-crosslinking was performed at 65 ◦C overnight with periodic 88

agitation. Recovered DNA was purified using the Qiagen PCR extraction kit. 5% 89

input and 2 µl of eluted DNA was used for quantitative rtPCR with gene-specific Q10 90

primers and SYBR green (BioRad 170-8880). For HEK293 cells, the EZ-CHIP assay 91

kit (Millipore 17-371)was used following themanufacturer’s instructions. Sonicated 92

chromatin was incubated with 2 µg of anti-Flag antibodies (Sigma F1804) or 93

normal mouse IgG (Santa Cruz sc-2025) at 4 ◦C overnight. Antibody–protein–DNA 94

complexes were recovered by the addition of salmon sperm DNA/protein G agarose 95

slurry (Millipore 16-201). Immunoprecipitated DNA fractions were de-crosslinked 96

at 65 ◦C overnight and purified using QIAquick columns (QIAGEN). Quantitative 97

rtPCR was performed as above except 1% input was used. All quantitative rtPCRs 98

were performed in triplicate using the following conditions: 1 cycle at 95 ◦C 5min, 99

followed by 20–40 cycles of 95 ◦C 15 s, 60–64 ◦C 30 s and 72 ◦C 30 s. 100

Immunoblot assays. Whole-cell lysates were prepared from lysis of HepG2 and 101

Rcho-1 trophoblast stem cells in a standard radioimmunoprecipitation buffer 102

containing a freshly diluted cocktail of protease and phosphatase inhibitors. For 103

preparation of liver lysates, frozen tissue (∼100mg) was thawed on ice followed 104

by homogenization in radioimmunoprecipitation buffer and then centrifugation 105

at maximum speed for 15min in 4 ◦C. Total protein concentration was measured 106

using the Bio-Rad Protein Assay kit (BioRad 500-0006). Denatured samples were 107

separated in 8–10% SDS–polyacrylamide gels, transferred onto PDVF membranes, 108

blocked in 5%milk–PBS solution and incubated overnight in 4 ◦Cwith the following 109

primary antibodies: anti-E2F3 1:5,000 (Santa Cruz sc-878x), anti-Flag 1:1,000 110

(Sigma F1804), anti-tubulin 1:5,000 (Sigma T6199) and anti-HSP70 1:5,000 (BD 111

Transduction Laboratories 610608). HRP-conjugated species-specific secondary 112

antibodies were applied to membranes followed by detection with the SuperSignal 113

West Femto kit (Thermo Scientific 34095). 114

Immunohistochemistry and immunofluoresence microscopy. After antigen 115

retrieval using Target Retrieval Solution (DAKO S1699), 5 µm deparaffinized 116

sections of placenta or liver tissues were incubated with primary antibodies at 117

4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against 118

residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD 119

Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin 120

A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 121

(Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental 122

lactogen 1:200 (PL-1, F. Talamantes). TGCs in M phase (Fig. 2h) were identified 123

by detecting P-H3 or the lineage-specific PL-1 protein. For immunofluorescence 124

signal detection, fluorophore-conjugated secondary antibodies were applied to the 125

slides. For immunohistochemistry, the VECTASTAINABC systemwas used (Vector 126

Laboratories). 127

BrdU incorporation assays. Pregnant females (E10.5) received a single intraperi- 128

toneal injection of BrdU (Sigma B5002) in sterile PBS at 100 µg g−1 body weight 1 h 129

before being euthanized for collection of placentae. Deparaffinized tissue sections of 130

5 µm were used for BrdU detection after incubation in 2N HCl and neutralization 131

with 10mM sodium borate (pH 8.0). Primary antibody was then applied overnight. 132

pIpC injections. Six-week-old mice received 5 intraperitoneal injections of 250 µg 133

of pIpC (Sigma P1530) dissolved in sterile PBS every alternate day. Mice were 134

euthanized 24 h after the last injection, and their livers were collected for flow 135

cytometry and microarray analyses. 136

NATURE CELL BIOLOGY

Leon05
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Leon05
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Q9: Edit as follows: "Optical sections with plane-to-plane resolution ...".
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Q10: "ChIP assays used 5% of input DNA as the standard for 'relative occupancy' and 2ul of eluted DNA from immunoprecipitated samples for quantitative rtPCR with gene-specific primers ...."
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DOI: 10.1038/ncb2595 METHODS

Carbon tetrachloride injection. Six-month-old mice received a single intraperi-1

toneal injection of 10% carbon tetrachloride (Sigma 319961) dissolved in corn oil.2

Mice were euthanized at 0, 24, 48, 60 and 168 h (when liver regeneration is complete3

in rodents58) post-injection. Mice were administered an intraperitoneal injection of4

BrdU solution (100 µg g−1 body weight) to label hepatocytes in S phase of the cell5

cycle 1 h before being euthanized.6

Tamoxifen induction of cre. Tamoxifen chow (Teklad Lab Animal Diets, Harlan7

Laboratories) was prepared by mixing 3 kg of irradiated standard chow with 1 g8

tamoxifen (Sigma T5648). For pre-weaning induction of cre, 7-day-old pups and9

theirmother were switched from regular to tamoxifen chow for 1 week. After 1 week,10

they were again fed regular chow until weaning and up to time of euthanization (age11

2 months). For post-weaning induction of cre, pups were weaned from their mother12

at age 21 days and maintained on tamoxifen chow starting from age 28 days up to13

age 2 months. Tamoxifen chow was stored in the dark at 4 ◦C and changed weekly.14

It was provided to mice at 40–80mg kg−1 body weight59.15

X-gal staining. OCT-embedded placentae were sectioned and fixed in 0.2%16

PBS–glutaraldehyde solution (1.25mMEGTA at pH 7.3 and 2mMMgCl2). Sections17

were rinsed with wash buffer (2mMMgCl2, 0.01% sodium deoxycholate and 0.02%18

IGEPAL CA-630 (Sigma I8896)) and stained in LacZ solution (4mM potassium19

ferricyanide, 4mM potassium ferrocyanide and 1mgml−1 X-gal in wash buffer)20

at 37 ◦C overnight with protection from light. Nuclear Fast red was used as a21

counterstain.22

E2F-binding-site analysis. Promoter sequences (−3000 bp ∼+2000 bp) of genes23

antagonistically regulated by E2F1–3 and E2F7/E2F8 were obtained from the24

UCSC genome browser (http://genome.ucsc.edu/). consensus E2F-binding sites25

were identified using TFSearch (http://www.cbrc.jp/research/db/TFSEARCH.html).Q11 26

Statistical analyses. Binucleated TGCs, as a percentage of a minimum of 143 total27

TGCs assessed in each genetic group, were quantified in confocal images using the28

LSM image browser (Zeiss). All other immunohistochemistry/immunofluorescence-29

microscopy-associated quantification of TGCs/hepatocytes was performed using30

Metamorph 6.1 in photomerged images (placenta) or 5–7 random images (liver)31

acquired at 40× from a minimum of three sections per sample. The results are32

reported as an average percentage± s.d. of positive cells in the number (n) ofQ12 33

control and mutant samples analysed. Flow cytometry profiles (for example, Fig. 3f)34

are representative of a minimum of three independent experiments per genotype35

and age group. Flow cytometry results are reported as an average percentage± s.d.

of nuclei with a certain DNA content out of a minimum of 30,000 nuclei gated 36

per sample in n control and mutant samples analysed. Pairwise comparisons 37

were evaluated by two-tailed Student’s t -tests, whereas multiple comparisons were 38

evaluated by one-way analysis of variance (ANOVA). When appropriate, P values 39

were adjusted by the Holm’s method within each ploidy category. 40

Accession numbers. The gene expression microarray data used in this study can 41

be found in GEO using accession numbers GSE32353, GSE32354 and GSE32355. 42

48. Hardie, D. C., Gregory, T. R. & Hebert, P. D. From pixels to picograms: a beginners’ 43

guide to genome quantification by Feulgen image analysis densitometry. J. 44

Histochem. Cytochem. 50, 735–749 (2002). 45

49. Mayhew, C. N. et al. Liver-specific pRB loss results in ectopic cell cycle entry and 46

aberrant ploidy. Cancer Res. 65, 4568–4577 (2005). 47Q1350. Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucl. Acids 48

Res. 31, e15 (2003). 49

51. Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A Comparison of 50

normalization methods for high density oligonucleotide array data based on bias 51

and variance. Bioinformatics 19, 185–193 (2003). 52

52. Yushkevich, P. A. et al. User-guided 3D active contour segmentation of anatomical 53

structures: significantly improved efficiency and reliability. NeuroImage 31, 54

1116–1128 (2006). 55

53. Zhu, S. C. & Yuille, A. Region competition: unifying snakes, region growing, 56

and bayes/mdl for multiband image segmentation. IEEE Trans. Pattern Anal. 18, 57

884–900 (1996). 58

54. Sahgal, N., Canham, L. N., Canham, B. & Soares, M. J. Rcho-1 trophoblast stem 59

cells: a model system for studying trophoblast cell differentiation.Methods Mol. Med. 60

121, 159–178 (2006). 61

55. Liu, F., Song, Y. K. & Liu, D. Hydrodynamic-based transfection in animal by systemic 62

administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999). 63

56. Ullah, Z., Kohn, M. J., Yagi, R., Vassilev, L. T. & DePamphilis, M. L. Differentiation 64

of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 65

activity. Gen. Dev. 22, 3204–3236 (2008). 66

57. Nagahama, H. et al. Spatial and temporal expression patterns of the cyclin- 67

dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse 68

development. Anat. Embryol. 203, 77–87 (2001). 69

58. Schultze, B., Gerhard, H. & Maurer, W. Liver Regeneration after Experimental Injury 70

(Grune and Stratton, 1975). 71

59. Kiermayer, C., Conrad, M., Schneider, M., Schmidt, J. & Brielmeier, M. Optimization 72

of spatiotemporal gene inactivation in mouse heart by oral application of tamoxifen 73

citrate. Genesis 45, 11–16 (2007). 74

NATURE CELL BIOLOGY

Leon05
Sticky Note
Q11: Amended text is fine, except "consensus" should be capitalized as it is the first word of the sentence.
Leon05
Sticky Note
Q12: Fine as amended
Leon05
Sticky Note
Nuc. Acids Res. Vol. 31, No. 4 e15, 1-8 (2003)
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Page 1

Query 1:Further information given in the corresponding

author address not included in affiliation 1. OK?

Page 2

Query 2:Should ‘independent’ be ‘independently’ here?

Query 3:‘novel’ changed to ‘new’ here, and deleted from

before ‘lineage-specific’ in the third sentence of thefirst paragraph, according to style. OK?Query 4:‘while’ changed to ‘and’ here. OK (or is, for example,

‘whereas’ more appropriate)?

Page 9

Query 5:Text amended to ‘APC (also known as the

cyclosome)’ here, according to style. Please check.

Page 10

Query 6:Text amended to ‘As loss of ... of TGCs and’ here.

OK?Query 7:Please provide affiliations for F. Talamantes, M.

Soares and S. Jacobs.Query 8:Please confirm statement: ‘The authors declare no

competing financial interests.’

Page 12

Query 9:So the text can be formatted appropriately, please

clarify what is meant by ‘between-plane-plane’.Query 10:Please amend the text here to avoid the sentence

starting with a number.

Page 13

Query 11:Text amended to ‘consensus E2F-binding sites’

here. Is this correct? And, also please ensurethat the hyphenation of ‘-binding’ is appropriatethroughout.

Query 12:Please check that the intended meaning of the text

‘The results are ... mutant samples analysed.’ has beenretained after editing.Query 13:Please provide page range/article id for ref. 50.

General Queries

Query 14:For the representation of gene symbols and geno-

types we follow the standard scientific conventionsand nomenclature found in databases such as HUGOfor humans, MGI for mice or Flybase for Drosophila.Accordingly, many changes may have been madethroughout the text and figures. Please check thatwe have interpreted each instance correctly.

Query 15:Please note that reference numbers are formatted

according to style in the text, so that any referencenumbers following a symbol or acronym are givenas ‘ref. XX’ on the line, whereas all other referencenumbers are given as superscripts.

Query 16:The ‘Control’ for figure 1d does not seem to be

defined in the caption. Please check.

Query 17:Please check sentence ‘Uncropped images...’ added

to the end of the captions of figures 2 and 5, andsupply Supplementary Fig. number

Query 18:Please check that the intended meaning of the

caption of figure 2 has been retained after editing.Also, the scale bar in 2j is not defined. Please amendcaption.

Query 19:The insets in figure 3a are not mentioned in the

caption—please amend caption. (And please checktext added to captions of 6a,7a to define those insets).

Query 20:Acronyms ‘F7’, ‘F8’ and ‘TS’ removed from text,

according to style, and the caption of figure 5amended so a single definition is given, and the termsare used in full throughout the rest of the caption.Please check that the intended meaning has been

Leon05
Sticky Note
Q14: OK
Leon05
Sticky Note
Q15: OK
Leon05
Sticky Note
Q16: I believe it is defined already; i.e. " Feulgen .... in E9.5 control and 123tko ..."
Leon05
Sticky Note
Q17: Uncropped images for genotyping gels presented in Fig. 2 (2l) were not requested by Editor, thus the sentence in the legend caption relating to this should be removed. Uncropped images for blots in Fig. 5 were provided to the Editor in a separate file as requested/per instructions (i.e. not in the Supplementary section). So it is not at all clear how this should be handled. Our suggestion would be to delete the sentence from the caption altogether or refer to online materials if indeed NCB plans to include these data online.
Leon05
Sticky Note
Q18: Caption of Fig 2 is ok as amended. Please amend/add the definition of scale bars in Fig. 2: "Scale bars, 10um (b), 12.5um (e,f), 5um (h), 10um (i), 10um (j, top panel), and 0.5um (j, bottom panel)."
Leon05
Sticky Note
Q19: Please add the following to Fig 3a: The lower right insets show high magnification view of randomly selected nuclei. The caption for Fig 6a is fine as amended. The caption for Fig. 7a should read: "The lower right insets show higher magnifications of the areas outlined in the main panels."
Leon05
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Q20: OK as amended
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retained.

Query 21:Can ‘∗P ≤ 0.05;’ be deleted from the end of the

caption of figure 7, as it does not seem to be usedin the figure.

Leon05
Sticky Note
Q21: Yes please delete "*P<0.05"