Fig. S1

A TCGA cohort

** ** *** ** ** 600 500 500 ) 400 ) 400 ) 400 300 300 RSEM (RSEM RSEM ( ( 200 200 200 E2F2 E2F2 E2F2 100 100

0 0 0 SL HCC T1 T2 T3 T4 I II III IV n = 50 n = 371 Tumor stage Disease stage

B C

Birth 14 day-old 1 month-old 9-month-old WT E2f2-/- E2F2 Vehicle Transferrin Dietylnitrosamine (DEN)

Chow diet Liver High-fat diet WT E2f1-/- E2f2-/-

D Cell cycle mRNAs CD WT CD E2f2-/- 3 #

) DEN-HFD WT # * # DEN-HFD E2f2-/-

units 2

* 1

Fold Change * ** Arbitrary * ( ** 0 Plk1 Ccna2 Ccne1 Ccnd3 Tk Orc1

E

Cell cycle mRNAs

3

) * HFD WT HFD E2f2-/-

units 2

1

Fold Change * Arbitrary ( * ** 0 Plk1 Ccna2 Ccne1 Ccnd3 Tk Orc1 A B Fig. S2 Downregulated pathways Lipid oxidation mRNAs

(9 month-old DEN-HFD E2f2-/- vs DEN-HFD WT) ) 2.0 *** # *** ** -log2 (Adjusted p value) 1.5 * *** * units ** * *** 0 10 20 30 40 50 1.0 ## Immune System (n=242) 0.5 Fold change Arbitrary

Signaling by Rho ( GTPases (n=87) 0.0 Platelet activation, signaling Rxra Acsm1 Acsm3 Acsl1 Acadl Acadsb and aggregation(n=58) Integrin cell surface Oxidative phosphorylation mRNAs interactions (n=24) ) 2.0 # Extracellular matrix *** *** organization (n=54) # * *** ** units 1.5 * * *** # Axon guidance (n=50) 1.0 *** # # # Cell Cycle (n=84) 0.5 Fold change Arbitrary

Non-integrin membrane-ECM ( interactions (n=13) 0.0 Immunoregulatory interactions Ndufs5 Ndufs7 Ndufv1 Sdha Cyb5a Atpif1 Atp5a1 between a Lymphoid and a non-Lymphoid cell (n=23) CD WT CD E2f2-/- DEN-HFD WT DEN-HFD E2f2-/-

C D Fatty acid β-oxidation (FAO) rate Oxidative phosphorylation Lipid oxidation mRNAs mRNAs ASM CO2 -/-

) 1.5 ** 2.0 HFD WT HFD E2f2 10000 * 80 * * hour * ** / 8000 1.5 ** ** 60 units 1.0 6000 1.0 liver 40 Palmitate 4000 0.5

C- 0.5

20 Fold change

14 2000 Arbitrary ( nmol/g 0 0 0.0 0.0 HFD WT HFD E2f2-/-

E

WT E2f2-/- WT E2f2-/- CD HFD DEN-HFD CD HFD DEN-HFD 1 2 1 2 Plk1 Ccna2 Cell cycle Ccne1 Ccnd3 Tk Orc1 1: HFD vs CD Cpt1 2: DEN-HFD vs HFD Cpt2 Acaa2 NS Ppara Ppargc1a Ppargc1b Lipid p<0.05 Rxra oxidation Acsm1 Acsm3 p<0.01 Acsl1 Acadl p<0.001 Acadsb Ndufs5 Ndufs7 Ndufv1 Oxidative Sdha phosphorylation Cyb5a Atpif1 Atp5a1

Intensity Significant (log2 fold change) differences 3 0 -3 Fig. S3 A E2F2 mRNA E2F2 mRNA

1.5 3 )

) ** siControl HepG2 pCMV HepG2

siE2F2 HepG2 units units 1.0 2 pE2F2 HepG2

0.5 *** 1 Fold change Fold change Arbitrary Arbitrary ( (

0.0 0

B C

Fatty acid β-oxidation (FAO) rate MCM3 mRNA Cell cycle

ASM CO2 pCMV HepG2 40 pCMV HepG2 2500 80 2.5 (%) )

hour pE2F2 HepG2 pE2F2 HepG2 hour ** * 2000 2.0 30

60 cells

** units

1.5 ** of 1500 20 / protein

/ protein 40

Palmitate 1.0 Palmitate 1000 C-

C- 10 /mg /mg Fold change /mg /mg 14 20 0.5 14 Arbitrary

500 ( 0.0 0 pmol Percentage pmol 0 0 G0/G1 S G2/M

D

E2f2 mRNA 10 ) AAV8-Gfp AAV8 8 **

AAV8-Gfp AAV8-E2f2 units AAV8-E2f2 6 E2F2 4 Fold change Arbitrary

GAPDH ( 2 0

E F

Fatty acid β-oxidation (FAO) rate Cell cycle mRNAs ASM CO2 2.5 AAV8-Gfp

8000 100 ) AAV8-Gfp 2.0 AAV8-E2f2 hour hour 80 / /

6000 units AAV8-E2f2 1.5 60

liver *** liver 4000 Palmitate Palmitate 1.0 40 C- C- Fold change 14 14

2000 Arbitrary 0.5 20 ( nmol/g nmol/g 0.0 0 0 Mcm3 Orc1 ccnd3 Plk1 A Cell cycle mRNAs Fig. S4

3 CD WT *** -/- ) # # CD E2f2 2 * DEN-HFD WT units DEN-HFD E2f2-/-

1 Fold change Arbitrary ( * * * * 0 Plk1 Ccna2 Ccne1 Ccnd3 Tk Orc1 Mcm3 Dhfr Rrm2

CD WT CD E2f2-/- DEN-HFD WT DEN-HFD E2f2-/-

B CD DEN-HFD Ki67 levels 2.5 CD WT -/- WT 2.0 CD E2f2 DEN-HFD WT 1.5 DEN-HFD E2f2-/-

of positive cells 1.0 /- - 0.5 E2f2 0.0 Percentage

C D Inflammation mRNAs Liver weight to body weight ALT 2.5 ## 80 5 ## WT ) 2.0 * 60 4 -/- E2f2 units 1.5 3 40 % *** U/L 1.0 *** 2 * ## Fold change Arbitrary 0.5 20 ### ( 1 # 0.0 0 0 CD HFD DEN-HFD CD HFD DEN-HFD Tnf-α Il1-β Il6 Adgre1 CD WT CD E2f2-/- DEN-HFD WT DEN-HFD E2f2-/- E WT E2f2-/- CD DEN-HFD CD DEN-HFD Protein levels CD WT peIF2-α (Ser 51) p=0.052 CD E2f2-/- 5 Transferrin # DEN-HFD WT DEN-HFD E2f2-/- pIRE1-α (Ser 724) 4

units) 3 * Transferrin * 2 ** ## Fold change Fold Arbitrry ## eIF2-α ( 1 ### GRP78 0 IRE1-α peIF2-α pIRE1-α eIF2-α IRE1-α GRP78

Transferrin A Fig. S5 Lipid oxidation mRNAs CD WT CD E2f2-/- ## 2.0 DEN-HFD WT ## ## DEN-HFD E2f2-/- 1.5 * * 1.0

Fold change 0.5 (Arbitrary units) (Arbitrary 0.0 Rxra Ppara Ppargc1b Cpt1 Acsm1 Acsm3 Acadl Acadsb Acaa2 FA Transcription factors FA activation β-oxidation chain reactions transporter B Oxidative phosphorylation mRNAs 2.0 # # ## 1.5 # * CD WT -/- 1.0 CD E2f2 DEN-HFD WT -/- Fold change 0.5 DEN-HFD E2f2 (Arbitrary units) (Arbitrary 0.0 Ndufs5 Ndufs7 Ndufv1 Sdha Cyb5a Atpif1 Atp5a1 C Lipid oxidation mRNAs 2.0 HFD WT ) * HFD E2f2-/- 1.5 * * units

1.0 * ** * * ** 0.5 Fold change Arbitrary ( 0.0

FA transporters Transcription FA activation β-oxidation factors chain reactions

D E Fatty acid β-oxidation (FAO) rate

Oxidative phosphorylation mRNAs ASM CO2 1.5 40

) 4000

*** hour hour * / / 3000 30 units 1.0 ** * liver liver 20

2000 Palmitate Palmitate

* C-

0.5 C- 14 Fold change 14 1000 10 Arbitrary nmol/g ( nmol/g 0.0 0 0 Ndufs5 Ndufs7 Ndufv1 Sdha Cyb5a Atpif1 Atp5a1

HFD WT HFD E2f2-/- HFD WT HFD E2f2-/- F Oxygen consumption rate (hepatocytes)

p=0.08 400 300 Rot&AA CD WT 300 200 Oligomycin CD E2f2-/- 200 FCCP /min/protein /min/protein 100 100 pmol pmol 0 0 0 15 30 45 60 75 90 Time (min) Fig. S6

A B Glycolysis (hepatocytes) Glycolysis (hepatocytes)

Glucose 60 60 50 2-DG 50 Glucose 2-DG 40 40 40 40 30 30 ) protein /min/ ) protein /min/ ) protein /min/ ) protein /min/ * 20 20

20 20 mpH mpH mpH mpH 10 10 0 0 0 0 ECAR ( ECAR ECAR ( ECAR ECAR ( ECAR ECAR ( ECAR 0 15 30 45 60 75 90 0 15 30 45 60 75 90 Time (min) Time (min)

-/- CD WT CD E2f2 DEN-HFD WT DEN-HFD E2f2-/-

C Glycolysis mRNAs in liver

2.5 ### CD WT 2.0 CD E2f2-/- DEN-HFD WT 1.5 *** ## DEN-HFD E2f2-/- 1.0 ***

Fold change 0.5 (Arbitrary units) (Arbitrary 0.0 Gck Pklr

D E F de novo lipogenesis mRNAs Malonyl-CoA de novo lipogenesis of TG

2.0 250 TG 80 ** 1.5 ** 200 liver 60 #

150 liver 1.0 into 40 CoA/µg protein CoA/µg

- 100

Fold change 0.5

dpm/g 20 (Arbitrary units) (Arbitrary 50 0.0 Acetate H- 0

0 3 Fasn Acaca Me2 Me3 G6pdx pg malonyl CD WT CD E2f2-/- DEN-HFD WT DEN-HFD E2f2-/-

G H

VLDL metabolism mRNAs CD WT Hepatic TG secretion rate CD WT CD E2f2-/- CD E2f2-/- 2.0 8 DEN-HFD WT DEN-HFD WT -/- -/- 1.5 DEN-HFD E2f2 6 DEN-HFD E2f2 hour

1.0 TG/ 4 Fold change 0.5 mM 2 (Arbitrary units) (Arbitrary

0.0 0 Apob Apoe Fig. S7 A B

de novo lipogenesis of TG de novo lipogenesis mRNAs 1.5 40 TG ) HFD WT HFD WT 30 -/- liver HFD E2f2-/- units 1.0 HFD E2f2 liver

into 20 0.5 dpm/g Fold change 10 Arbitrary ( Acetate Acetate H-

0.0 3 0 Fasn Acaca G6pdx

C D

VLDL metabolism mRNAs Hepatic TG secretion rate 1.5 * HFD WT 4 HFD WT HFD E2f2-/- HFD E2f2-/- 3 1.0 hour 2 TG/ 0.5 Fold change

mM 1 (Arbitrary units) (Arbitrary

0.0 0 Apoe Apob Fig. S8 A B E2f2 mRNA (hepatocytes) Cpt2 mRNA (hepatocytes)

600 1.5 ) *** ) Ad-Gfp Ad-E2f2 Ad-Gfp Ad-Gfp Ad-E2f2 units Ad-E2f2 400 units 1.0 E2F2 *** 200 0.5 Fold change Fold change

GAPDH Arbitrary Arbitrary ( ( 0 0.0

C Ad-E2f2 ChIP (hepatocytes) 6 Fatty acid metabolism Ad-Gfp

IgG 4 Ad-E2f2 ver enrichment * o 2 Fold

0 Genes: Cpt2 Cpt1 Acaca Fasn Scd1 Srebp1c

Fatty acid oxidation Fatty acid synthesis

D E

Ad-E2f2 ChIP (hepatocytes) Ad-E2f2 ChIP (hepatocytes) Glycolysis Mitochondria 6 * 2.5 Ad-Gfp Ad-E2f2 2.0 Ad-Gfp 4 1.5 Ad-E2f2 IgG IgG ** * ver

enrichment 1.0

ver 2 enrichment o o 0.5 Fold Fold 0 0.0 Genes: Gck Pklr Pdk4 Chrebp Genes: Sdha Tfam Ucp2 A pRB protein levels Rb mRNA Fig. S9 Hepatocytes 1.5 1.5 siControl siControl siRb 1.0 1.0 siRb pRB *** 0.5 0.5 * Fold change (Arbitrary units) (Arbitrary GAPDH Fold change (Arbitrary units) (Arbitrary 0.0 0.0 B

Cpt2 mRNA levels from hepatocytes 1.5 1.5 siControl 1.0 1.0 siRb *

0.5 0.5 Fold change Fold change (Arbitrary units) (Arbitrary (Arbitrary units) (Arbitrary 0.0 0.0 DEN-HFD WT CD WT C

CPT2 promoter

CREB binding site

D Protein levels in liver Fatty acid β-oxidation rate

DEN-HFD WT 1.5 1.5 CO 15000 ASM 800 2 Control ASO CREB ASO Control ASO 600 1.0 1.0 CREB ASO CREB1 10000 * * 400 Transferrin 0.5 0.5 5000 CPT2 Fold change *** 200 (Arbitrary units) (Arbitrary nmol/g liver/hour nmol/g liver/hour Transferrin 0 0 CREB1 CPT2 0 0 DEN-HFD WT DEN-HFD WT E Protein levels in liver Fatty acid β-oxidation rate

ASM CO2 1.5 1000 DEN-HFD E2f2-/- 2.0 15000 800 Control ASO Control ASO CREB ASO 1.5 1.0 CREB ASO CREB1 10000 600 1.0 Transferrin 400 0.5 *** 5000

Fold change 0.5 CPT2 200 Transferrin units) (Arbitrary nmol/g liver/hour 0 0 nmol/g liver/hour 0 CREB1 CPT2 0 DEN-HFD E2f2-/- DEN-HFD E2f2-/- A TCGA cohort Fig. S10 ** *** * *** * 9000 9000 9000 8000

8500 ) )

) 4000 4000 8000 4000 3000 RSEM

3000 ( RSEM RSEM ( ( 3000 2000 2000 2000 E2F1 E2F1 E2F1 1000 1000 1000 0 0 0 SL HCC T1 T2 T3 T4 I II III IV n = 50 n = 371 Tumor stage Disease stage

B Upregulated pathways C Upregulated pathways (9 month-old DEN-HFD E2f1-/- vs DEN-HFD WT) (9 month-old DEN-HFD E2f1-/- vs DEN-HFD WT)

-log2 (Adjusted p value) -log2 (Adjusted p value) 0 50 100 150 200 0 10 20 30 Metabolism (n=291) Platelet activation, signaling Biological oxidations (n=55) and aggregation (n=53) Metabolism of amino acids and derivatives (n=61) Axon guidance (n=54) Metabolism of lipids (n=98) Immune System (n=202) TCA cycle and respiratory electron transport (n=34) Synthesis of bile acids and bile salts via (n=11) RHO GTPases Activate Formins (n=30) Metabolism of vitamins and cofactors (n=35) Cell surface interactions at the vascular wall (n=25) Phase II - Conjugation of compounds (n=22) Peroxisomal lipid metabolism (n=12) Integrin cell surface interactions (n=19) Respiratory electron transport (n=17) Extracellular matrix organization (n=46) Mitochondrial Fatty Acid Beta-Oxidation (n=13) Branched-chain amino acid catabolism (n=9) Cell Cycle (n=78) Lysine catabolism (n=7) Signal Transduction(n=253) Post-translational protein modification (n=50) Mitotic Prometaphase (n=34) Glyoxylate metabolism and glycine degradation (n=9) Tyrosine catabolism (n=4) Resolution of Sister Chromatid Cohesion(n=24) Arachidonic acid metabolism (n=13) Signaling by VEGF (n=21) Sulfur amino acid metabolism (n=8) Class I peroxisomal membrane protein import (n=7) Ephrin signaling (n=8) alpha-linolenic acid (ALA) metabolism (n=6)

D Oxidative phosphorylation mRNAs CD WT

) 2.0 ## -/- *** *** CD E2f1 1.5 * ** * ** units *** *** DEN-HFD WT 1.0 ** ## DEN-HFD E2f1-/- # 0.5 Fold change Arbitrary ( 0.0 Ndufs5 Ndufs7 Ndufv1 Sdha Cyb5a Atpif1 Atp5a1

E Lipid oxidation mRNAs CD WT ) 2.0 * # CD E2f1-/- units 1.5 * * * DEN-HFD WT 1.0 # * ### DEN-HFD E2f1-/-

Fold change 0.5 Arbitrary ( 0.0 Rxra Acsm1 Acsm3 Acsl1 Acadl Acadsb Transcription β-oxidation FA activation factor chain reactions F G 3 month-old E2f1 mRNA 9 month-old E2f1 mRNA )

) p=0.076 10 # WT 6 ## WT 8 -/- E2f2-/-

E2f2 units

units ## ### 6 4 * * ## 4 # ## 2 *** 2 * ##

# Fold change Fold change Arbitrary Arbitrary 0 ( ( 0 CD HFD DEN-HFD CD HFD CD HFD DEN