Cell Host & Microbe, Volume 26

Supplemental Information

The Nuclear Matrix Protein SAFA Surveils

Viral RNA and Facilitates Immunity by Activating

Antiviral Enhancers and Super-enhancers

Lili Cao, Shengde Liu, Yunfei Li, Guang Yang, Yujie Luo, Siji Li, Hongqiang Du, YingchiZhao, Dandan Wang, Jingxuan Chen, Zeming Zhang, Mo Li, Songying Ouyang, XiangGao, Yujie Sun, Zekun Wang, Long Yang, Rongtuan Lin, Penghua Wang, and Fuping You

Supplemental Information

Supplemental Figures

Figure S1. SAFA is critical for antiviral immune responses. Related to Figure 1.

Figure S2. SAFA mediates antiviral immunity in vivo. Related to Figure 2.

Figure S3. Oligomerized SAFA mediates IFNβ transcription. Related to Figure 3.

Figure S4. SAFA senses the nuclear viral RNA. Related to Figure 4.

Figure S5. SAFA facilitates antiviral immunity through interacting with TOP1 and

SMARCA5. Related to Figure 5.

Figure S6. SAFA activates the distal enhancers of type I IFN. Related to Figure 6.

Figure S7. Super-enhancers were activated by viral infection. Related to Figure 7.

Figure S8. SAFA associated super-enhancers potentiate antiviral responses. Related to

Figure 7.

Figure S9. SAFA facilitates extra-nuclear antiviral signaling. Related to Figure 7.

Supplemental Videos

Video1: Movie of Safafl/fl mice.

Video2: Movie of Safafl/fl Lyz2 Cre mice.

Video3: Movie of Safafl/fl mice post i.v. infection with HSV-1.

Video4: Movie of Safafl/fl Lyz2 Cre mice post i.v. infection with HSV-1.

Supplemental Tables

Table S1: Primers and guide RNA sequence used in this study. Related to STAR Methods.

─/──/─

B

he

art

thym

us

sp

lee

n

lun

g

kid

ne

y

sto

ma

ch

bra

in

live

r

ova

ry

SAFA

Tubulin

inte

stin

e

L9

29

EL

4

DC

4.2

iBM

DM

ME

F

29

3T

PC

3

A3

75

He

pG

2

HC

T11

6

U2

OS

HT

10

80

Hela

SAFA

Tubulin

C

A

FE

G

Mock VSV HSV-1

WT

iB

MD

M

Sa

fa–

/–iB

MD

M

SAFA

Tubulin

WT

29

3T

SA

FA

–/–

29

3T

Scra

mb

le s

iRN

A

SA

FA

siR

NA

SAFA

Histone

D

H

J

SAFA

Tubulin

IFN-β (U/ml) 0 10 50 100 200

M

N

Tubulin

HnrnpH2

PO

Tubulin

HnrnpL

I K

L

─/─

Figure S1. SAFA is critical for antiviral immune responses. Related to Figure 1. (A) Functional screening for the genes that activate IFNβ

during virus infection. BMDM were infected with VSV. The cells were harvested at 6 hours after infection. Total RNA was isolated and reverse

transcribed to cDNA with SMART(er) cDNA synthesis (Clontech). Approximately 9,000 mouse full-length cDNAs cloned into pEXP-Lib vector

(Clontech) by using In-Fusion SMARTer Directional cDNA Library Construction Kit (Clontech). To identify cDNAs that encode protein that

enhances IFNβ-luciferase reporter activity during viral infection, 150 ng of empty vector, ELF4 cDNA positive control expression plasmids or

BMDM cDNA and 50 ng of IFNβ reporter were transfected into HEK293 cells.24 hours later, these cells were infected with VSV. Another 24 hours

later, luciferase activity was measured. This cDNA library was divided into ∼100 clones per pool and the plasmid DNA was prepared for IFNβ

luciferase reporter. The positive pools were further divided into subpools of ∼10 clones, which were tested for their ability to activate IFNβ in

reporter assays. Single clones were isolated from the positive subpools and again tested for their ability to activate IFNβ in reporter assays. The

positive clones were sequenced and identified by BLAST searches of the GenBank databases. (B) Immunoblotting showing the protein levels of

SAFA in different cell lines (upper) and different murine tissues (below). (C) Luciferase activity of IFNβ in HEK293 cells expressing IFNβ–Luci

plasmid and either an empty vector or SAFA plasmid, after 24 hours infected with VSV or HSV-1. (D) HEK293 cells were transfected with SAFA

siRNA or scramble siRNA, 36 hours later infected with HSV-1, HSV-1 mRNA was analysed by qRT-PCR (left), IFNβ–Luci activity was measured

(middle), and the knockdown efficiency was detected by immunoblotting (right). (E) Wild-type and SAFA–/– HEK293 cells were infected with VSV

or HSV-1, type I IFNs were measured by bioassay. Immunoblotting showing the expression of SAFA in HEK293 cells or iBMDMs after CRISPR-

Cas9 gene editing. (F) Wild-type and SAFA–/– HeLa cells were infected with VSV or HSV-1, IFNB1 mRNA was analysed by qRT-PCR. (G)

Luciferase activity of IFNβ in HEK293 cells expressing IFNβ–Luc plasmid and either an empty vector or SAFA plasmid, after 24 hours transfected

with poly (I:C) (left). Luciferase activity of IFNβ in wild-type and SAFA–/– HEK293 cells expressing IFNβ–Luci plasmid, after 24 hours infected with

VSV or transfected with poly (I:C) (right). (H) 2fTGH-ISRE Luci cells (left) or HT1080 cells transfected with SAFA-Luci plasmid for 24 hours (right)

cultured with medium containing type I interferon, and 6 hours later luciferase activity was measured. (I) Peritoneal macrophages were treated

with type I IFN, 24 hours later, and Safa and Isg15 mRNA were measured by qRT-PCR (J) MEF cells were treated with indicated dose of type I

IFN, 24 hours later, and Safa, Cxcl10, Isg15 and cGAS mRNA were measured by qRT-PCR. (K) MEF cells were treated with indicated dose of

type I IFN, 24 hours later, indicated protein levels were detected by immunoblotting. (L) MEF cells were infected with VSV, 24 hours later, Safa

mRNA were measured by qRT-PCR. (M) Wild-type, Elf4–/–, Mavs–/– and Sting–/– peritoneal macrophages were infected with VSV, 24 hours later

cells were harvested for ChIP with H3K27Ac antibody. ChIP-qRT-PCR experiments showing H3K27Ac binding to Safa enhancer activity. (N)

Luciferase activity of SAFA in HEK293 cells expressing indicated plasmids. (O and P) Wild-type, Hnrnpl–/– (O) and HnrnpH2 –/– (P) iBMDMs were

infected with VSV or HSV-1, 6 hours later, Ifnb1 transcript was measured by qRT-PCR. Immunoblotting show the expression of HNRNPL and

HNRNPH2 in iBMDMs after CRISPR-Cas9 gene editing.

14.5kb

Protein coding Loxp site

Safafl/fl

Safafl/fl

Lyz2-Cre

B

A C

D

Safa-loxtF/R

oIMR3066/67

oIMR3067/68

Safa MUS-F/R

MSa

faw

t/w

t -C

re+

/－

Sa

fafl/f

l-C

re+

/－

Sa

fafl/w

t -C

re–

/–

500bp

2000bpBMDM

Tail

WT

BMDM

WT

Sa

fa─

/─

Peritoneal macrophage

SAFA

Tubulin

Sa

fa─

/─

Figure S2. SAFA mediates antiviral immunity in vivo. Related to Figure 2. (A) Image depicting gene editing model for Safafl/fl-Lyz2 Cre mice. (B)

RT-PCR showing genotyping results of Safafl/fl-Lyz2 Cre mice. Primers SafaMUS-F/R flank the two loxp sites. Primers Safa-loxtF/R type for Safa flox

mice. Primers oIMR3066/67 and oIMR3067/68 type for Lyz2-Cre mice (left); immunoblotting showing the knockdown efficiency of SAFA in BMDMs

or peritoneal macrophages of Safafl/fl-Lyz2 Cre mice. (C) Age- and sex-matched Safafl/fl and Safafl/fl -Lyz2 Cre mice (n = 6 ) inoculated i.v. with HSV-1at 5×107 pfu per mouse, and liver were retrieved 5 days later for qRT-PCR analysis of HSV-1 genomic DNA. (D) qRT-PCR analysis of Cxcl10 mRNA

in wild-type and Safa–/– peritoneal macrophages infection with HSV-1 for 6 hours. (E and F) Age- and sex-matched Safafl/fl and Safafl/fl -Lyz2 Cre

mice (n=6) inoculated (i.v.) with VSV and the survival rates of mice were observed and recorded (E), and footprint analysis were did on day 5 after

infection (F). (G) Age- and sex-matched Safafl/fl and Safafl/fl -Lyz2 Cre mice (n=6) inoculated (i.v.) with VSV per mouse. 1 day after infection Ifnb1,

and Ifna1 in the blood were assessed by qRT-PCR. (H) ) Age- and sex-matched Safafl/fl and Safafl/fl -Lyz2 Cre mice were treated with anti-IFNAR

antibody to mimic deficiency of IFNAR and infected mice with HSV-1 (i.v.), and the survival rates of mice were observed and recorded. (I)&(J) IFNAR

iMEFs were infected with VSV (MOI=0.1) or HSV-1 (MOI=1), viral load and mRNA were measured by plaque assay and qRT-PCR. (K) Footprint

analysis on age- and sex-matched Safafl/fl and Safafl/fl -Lyz2 Cre mice with mock treatment.

Safa fl/fl-Lyz2 CreSafa fl/fl

E

Safa fl/fl-Lyz2 CreSafa fl/fl

F G H

I

KJ

A

Figure S3. Oligomerized SAFA mediates IFNβ transcription. Related to Figure 3. (A). HEK293 cells were transfected with Flag-SAFA and

HA-SAFA. 24 hours later, these cells were infected with HSV-1. Cell lysates were prepared and immunoprecipitaed with anti-Flag antibody at

6 hours after infection. (B) MEF cells were infected with HSV-1, VSV or SeV for 6 hours or transfected with ploy (IC) or Ct DNA (Calf thymus

DNA) for 24 hours, then stained with anti-SAFA antibody (red) and DAPI (blue), and imaged by confocal microscopy. (C) MEF cells were

infected with VSV or HSV-1 for 4 hours, then stained with anti-SAFA antibody (red) and DAPI (blue), and imaged by confocal microscopy.

Protein level of SAFA was detected by immunoblotting. Histogram shows quantification of indicated confocal signals. (D) HEK293 cells were

transfected with indicated plasmids, and 24 hours later indicated protein levels were detected by immunoblotting (left). HEK293 cells were

transfected with indicated plasmids, and 24 hours later cells were lysed into nuclear–cytoplasmic fractionations, and the cell localization of

indicated proteins were analyzed by immunoblotting (right).

HA-SAFA

Flag-SAFA

- + ++ + +

HSV-1 - - +

WCLFlag

HA

IPFlag

HA

SAFA DAPI Merge

Mo

ck

HS

V-1

VS

VP

oly

(I:C

)C

t D

NA

Se

V

10μm

B

SA

FA

De

l S

AP

De

l S

PR

Y

De

l AA

A+

De

l R

GG

Wa

lke

r A

mu

tan

t

Wa

lke

r B

mu

tan

t

Flag

Tubulin

Lamin B

Tubulin

Flag

SA

FA

De

l S

AP

De

l S

PR

Y

De

l AA

A+

De

l R

GG

Walk

er A

muta

nt

Wa

lke

r B

mu

tan

t

SA

FA

De

l S

AP

De

l S

PR

Y

De

l AA

A+

De

l R

GG

Walk

er A

muta

nt

Wa

lke

r B

mu

tan

t

Nuclear Cytoplasm

D

C

Lamin B

SAFA

Mo

ck

VS

V

HS

V-1

VS

VH

SV

-1M

ock

SAFA DAPI Merge

50μm

VSV RNA

VSV

IP:

IgG

SA

FA

SAFA

Input

- + + - + +

IgG

BA MergeDAPIdsRNA

Mo

ck

VS

VP

oly

(I:C

)

20μm

F

D

C

0

200

400

600

800

1000

0 20 40 60 80 100 120 140 160

SAFA RIP-Seq Reads depth of HSV-1 genome

(Kb)

Dep

th

Reference position

E

US10 US1 UL50

Base-pair probabilities

0 1

UL11

J

Tubulin

Lamin B

K

N

M

OMock Leader RNA

Ds R

NA

DA

PI

Me

rge

20μm

I

Mo

ck

HS

V-1

Merge DAPI SAFA dsRNA

10μm

P7

.1-h

SF

-hA

DA

R1

cd

P1

4-h

SF

3H

A-h

SA

FA

3F

-hS

AF

A

-AD

AR

1cd

Me

rge

20μm

L

HS

V-1

HS

V-1

+R

na

se

III

Mo

ck

VS

V

VS

V+

Rn

ase

III

IB：SAFA

IB: SAFA

IB: Tubulin

Native Page

SDS Page

H

(h)

F

(h)

nucleus cytoplasm

LaminB

GAPDH

HSV-1 0h 0h 9h 9h

G

Figure S4. SAFA senses the nuclear viral RNA. Related to Figure 4. (A) Hela cells were infected with VSV for 6 hours or transfected with

poly (I:C) for 24 hours, stained with J2 anti-dsRNA antibody (red) and DAPI (blue), and imaged by confocal microscopy. (B) HEK293 cells

were infected with VSV, 6 hours later cells were lysed, SAFA proteins were precipitated and the associated RNA isolated for RT-PCR (left) or

qRT-PCR (right), and immunoprecipitation efficiency were analyzed by immunoblotting (left). (C) Reads depth of HSV-1 genome of the SAFA

RIP-seq. (D)Bar and curve graph showing transcript abundance of HSV-1 RNA in HEK293 cells at 8 hours after HSV-1 infection and HSV-1

RNA affinity with SAFA in RIP-seq, (E) RNA structures predicted by RNAfold based on the prioritized HSV-1 RNA sequences binding with

SAFA. (F)&(G). HEK293 cells were infected with HSV-1 for the indicated time followed by cellular fraction. HSV-1 RNA was measured by

qRT-PCR, and immunoprecipitation efficiency were analyzed by immunoblotting. (H) HEK293 cells were infected with VSV or HSV-1 for 6

hours, cell lysates were prepared without or with Rnase III treatment for 15 minutes at room temperature, and resolved by native PAGE. (I)

MEF cells were infected with HSV-1 for 6 hours, stained with J2 anti-dsRNA antibody (red), anti-SAFA (green) and DAPI (blue), and imaged

by confocal microscopy. (J) Hela cells were infected with HSV-1, 6 hours later cells were lysed into nuclear–cytoplasmic fractionations, SAFA

proteins were precipitated and the associated RNA were isolated for qRT-PCR , and the cell localization of indicated proteins were analyzed

by immunoblotting. (K) Wild type and SAFA deficient peritoneal macrophages were treated with LPS for 6 hours, and Ifnb1 mRNA were

measured by qRT-PCR. (L) Expression of SAFA in stable cell lines were examined by western blotting and immunofluorescence. (M). Hela

cells were infected with VSV, 2 hours later cells were lysed into nuclear–cytoplasmic fractionations, and the abundance of VSV leader RNA

were measured by qRT-PCR. (L) Wild-type and MAVS-/- HEK293 cells were transfected with IFNβ–Luci plasmid and indicated plasmids,

then with or without VSV leader RNA plasmid (LD RNA) transfection, and 24 hours later the luciferase activity was measured. (O) Hela cells

were transfected with VSV leader RNA plasmid for 24 hours, stained with J2 anti-dsRNA antibody (red) and DAPI (blue), and imaged byconfocal microscopy.

A B CHEK293 cell

Cell lysis

Immunoprecipitate

with IgG or anti-SAFA

antibody

Mass Spectrometry

Mock VSV

D

treatment Mock Mock VSV

antibody IgG SAFA SAFA

SMARCA5

no-detectable no-detectable

TEQEEDEELLTESSKRTEQEEDEELLTESSK

VLIFSQMTRSVCLIGDKEQR

TOP1 no-detectableQPEDDLFDRAEEVATFFAK

QIALGTSK

DEDDADYKPKCDFTQMSQYFK

E

HA-Del SAP

HA-SAFA

Flag-SMARCA5

－ ＋ － － － －

WCL

＋ ＋ ＋ ＋ ＋ ＋

－ － ＋ － － －

HA-Del AAA+HA-Del SPRY － － － ＋ － －

－ － － － ＋ －

HA-Del RGG － － － － － ＋

IB:HA

IB:FlagIP:Flag

IB:HA

IB:Flag

HA-Del SAP

HA-SAFA

Flag-TOP1

－ ＋ － － － －

WCL

＋ ＋ ＋ ＋ ＋ ＋

－ － ＋ － － －

HA-Del AAA+HA-Del SPRY － － － ＋ － －

－ － － － ＋ －

HA-Del RGG － － － － － ＋

IB:HA

IB:FlagIP:Flag

IB:HA

IB:Flag

Flag-SMARCA5

SAFA － ＋

＋ ＋

Flag-pull down

input

Flag

SAFA

Flag

SAFA

Flag-TOP1

SAFA － ＋

＋ ＋

Flag-pull down

input

Flag

SAFA

Flag

SAFA

F G

20μm

Cd38

Nr4a1

Ccrn4l

Irf1

Nfkbiz

Sod2

Pim1

Socs3

Tnfaip3

Egr1

Junb

Marcksl1

Bcl3

Icam1

Zfp36

Tnfsf9

Nfkbia

Fosb

Egr2

Fos

Nfkbid

Ptgs2

Tlr2

Cxcl1

Cxcl2

Tnf

SWI/SNF – Independent

CpG – Island Promoters

WT Safa–/–

HSV-1 － ＋ － ＋

Ccrl2

Traf1

Cxcl11

Clec4e

Il1a

Csf2

Il23a

Ccl3

Gbp2

Gbp1

Il1b

SWI/SNF – Independent

Non CpG – Island Promoters

WT Safa–/–

HSV-1 － ＋ － ＋

Primary Response Genes

Map3k8

Serpine1

Arhgef3

Vcam1

Saa3

Ccl2

Il10

Ikbke

SWI/SNF – Dependent

IRF3 - Independent

WT Safa–/–

HSV-1 － ＋ － ＋

Actin

Peli1

Ifit2

Cxcl10

Irg1

Ifit1

Ifnb1

Ccl12

Mmp13

Ccl5

Ifit3

Actin

Actin

Tyk1

Rsad2

SWI/SNF – Independent

Secondary Response Genes

Irf7

Il6

Il12b

Nos2

Lcn2

Marco

Mx2

Serpinb3b

Mx1

H28

SWI/SNF – Dependent

IFNa4

Actin

WT Safa–/–

HSV-1 － ＋ － ＋

WT Safa–/–

HSV-1 － ＋ － ＋

Actin

H

Figure S5. SAFA facilitates antiviral immunity through interacting with TOP1 and SMARCA5. Related to Figure 5. (A) Diagram detailing

the process and results of mass spectrometry to find proteins interact with SAFA. (B) HEK293 cells were transfected with indicated plasmids

for 24 hours, then cells were lysed and coimmunoprecipitation analysis were did to detected the interaction between Flag-SMARCA5 and

indicated truncations of HA-SAFA. (C) HEK293 cells were transfected with indicated plasmids for 24 hours, then cells were lysed and

coimmunoprecipitation analysis were did to detected the interaction between Flag-TOP1 and indicated truncations of HA-SAFA. (D) Theindicated Flag-tagged proteins were isolated and purified with 3×Flag peptide, then incubated with SAFA protein, and pull-down using FLAG

antibody. (E) Wild-type and SMARCA5–/– HEK293 cells were transfected with IFNβ–Luci plasmid, 24 hours later infected with VSV or HSV-1,

and 24 hours later luciferase activity was measured. (F) Wild-type and TOP1–/– HEK293 cells were transfected with IFNβ–Luci plasmid, 24

hours later infected with or VSV, and 24 hours later luciferase activity was measured. (G) THP-1 cells were transfected with empty vector or

SAFA plasmids, after 24 hours infected with HSV-1, 8 hours later treated with DMSO or TOP1 inhibitor Camptothecin (1uM), another 8 hours

later IFNB1 mRNA was detected by qRT-PCR. (H) Wild-type and Safa–/– BMDMs were infected with HSV-1 for 6 hours, and indicated genes

mRNA level were detected by RT-PCR.

A

B

C

E

GSM940911 BMDM

GSM1022299 BMDM

GSM1356212 BMDM

GSM1356215 BMDM

GSM1634798 BMDM

GSM2425246 BMDM

GSM2241205 BMDM

GSM1183990 PMC

MUS

GSM785500

MacrophageGSM1327357

MacrophageGSM1327358

MacrophageGSM1327360

Macrophage

GSM1625978

Macrophage

GSM1587925 MV4-11

GSM1513830 MV4-11

GSM2108039 MV4-11

GSM2544239 THP-1

HOMO

F

G

D

Figure S6. SAFA activates the distal enhancers of type I IFN. Related to Figure 6. (A) Normalized ChIP-seq profiles showing regions of 3

putative Ifnb1 enhancers in mouse cells and 4 putative IFNB1 enhancers in human ceells with H3K27Ac antibody from Dataset Browser

(http://cistrome.org/db/#/). (B) Diagram detailing predicted enhancer position around type I interferon and CRISPR-Cas9 system mediated

deletion of predicted enhancers. The position of DNA binding motifs of SAR domain was marked by red dots. (C) Wild-type and Enhanceres–

/– HEK293 cells were infected with HSV-1. 24 hours later, the viral load was analyzed by plaque assay. (D) Diagram of the IFNB1-Promoter-

Enhancer-Luci plasmids. (E) HEK293 cells were transfected with wild type Promoter-Enhancer-Luci plasmids or the mutant lacking SAR in

the enhancer, after 24 hours infected with VSV. (F) Luciferase activity of indicated reporter plasmid in wild-type and SAFA–/– HEK293 cells

transfected with indicated plasmids, with HSV-1 infection for 24 hours. (G) ChIP-qRT-PCR experiments showing IRF3 and IRF7 binding to

IFNB promoter or enhancers depending on SAFA. Wild-type and SAFA–/– Hela cells were transfected without or with Flag-tagged IRF3 or

IRF7 plasmids, and 24 hours later with HSV-1 infection for indicated times, then cells were harvested for ChIP with Flag antibody.

CBA DHSV-1 MockVSV Mock HSV-1 MockVSV Mock

Low High

Stx

7E

if3

bS

qstm

1R

rag

cR

nf2

13

Bd

p1

Bckd

hb

Pp

p2

r5d

Cts

kA

4g

alt

24

10

00

4B

18

Rik

Wd

r45

bB

asp

12

21

04

08

F2

1R

ikT

xn

1N

r1h

2P

sm

d7

Ub

lcp

1C

tsk

No

ct

Ba

tfP

rrc2

cP

isd

Rp

s2

7l

Rip

k2

CT

03

01

61

.2T

or3

aA

rap

1S

lco

3a

1B

cd

in3

dM

orc

34

93

04

40

I19

Rik

Pkp

2B

rd2

Pie

zo

1T

cir

g1

Ha

t1S

lam

f9S

ca

rf1

Sig

lec1

Ptk

2b

Ed

em

1A

csl5

Ca

mta

2R

ab

2a

Clic

4V

asp

Gp

r84

Ssr2

Nrg

4F

yco

1T

me

m1

06

aA

tp1

3a

1B

cl2

a1

aC

lec2

dG

nb

1A

kt3

Sp

ata

13

Va

pa

Nd

ufs

2C

AA

A0

11

80

11

1.2

Te

sk1

Ccd

c8

8b

Gm

44

41

8E

hd

1Ir

ak2

Ncka

p1

l

1

10

100

1000

10000MockHSV-1

FPKM

Stx

7E

if3

bS

qstm

1R

rag

cR

nf2

13

Bd

p1

Bckd

hb

Pp

p2

r5d

Cts

kA

4g

alt

24

10

00

4B

18

Rik

Wd

r45

bB

asp

12

21

04

08

F2

1R

ikT

xn

1N

r1h

2P

sm

d7

Ub

lcp

1C

tsk

No

ct

Ba

tfP

rrc2

cP

isd

Rp

s2

7l

Rip

k2

CT

03

01

61

.2T

or3

aA

rap

1S

lco

3a

1B

cd

in3

dM

orc

34

93

04

40

I19

Rik

Pkp

2B

rd2

Pie

zo

1T

cir

g1

Ha

t1S

lam

f9S

ca

rf1

Sig

lec1

Ptk

2b

Ed

em

1A

csl5

Ca

mta

2R

ab

2a

Clic

4V

asp

Gp

r84

Ssr2

Nrg

4F

yco

1T

me

m1

06

aA

tp1

3a

1B

cl2

a1

aC

lec2

dG

nb

1A

kt3

Sp

ata

13

Va

pa

Nd

ufs

2C

AA

A0

11

80

11

1.2

Te

sk1

Ccd

c8

8b

Gm

44

41

8E

hd

1Ir

ak2

Ncka

p1

l

1

10

100

1000

10000WTSafa－/－

FPKM

E

F

18 20 22 24 26 28

Enrichr Reactome

Combined score

Innate Immune System

Signaling by the B Cell Receptor

Interleukin-1 signaling

Signaling by Interleukins

Cytokine Signaling in Immune System

Toll-Like Receptors Cascades

Downstream signaling events of B cell Receptor

Activated super-enhancers related genes after virus infection

Detoxification of Reactive Oxygen Species

CREB phosphorylation through the activation

Metabolism of nucleotides

Pyrimidine metabolism

MAP2K and MAPK activation

Post NMDA receptor activation events

Rho GTPase cycle

Transmission across Chemical Synapses

Enrichr Reactome

Combined Score5 7 9 11 13

5 7 9 11 13

Repressed super-enhancers related genes after virus infection

G

Moc

kVSV

0.0

0.5

1.0

1.5

2.0

2.5Pkp2

*

Moc

kVSV

0.0

0.5

1.0

1.5

2.0Slco3a1

*

Moc

kVSV

0

1

2

3

4

5Rftn1

*

Mrp

s2

1R

nf1

67

Ccm

2R

mn

d5

bK

cn

j2D

ock1

0T

np

o1

Yip

f2N

ed

d4

lP

atl1

Pfk

fb4

Ssp

nB

az1

bT

me

m6

0F

blim

1C

am

k1

Me

ttl2

Ub

l3In

o8

0e

Nd

ufa

13

Tb

l1xr1

Am

pd

2U

gt1

a7

c3

11

00

56

K0

7R

ikT

mco

1V

ars

18

10

04

3G

02

Rik

Tcta

Hd

ac4

Mrp

l33

Tlr

9T

tc7

Aste

1A

ldh

3b

1C

ea

ca

m1

9S

nh

g1

5Z

miz

1H

int1

Elo

vl5

Slc

35

a3

Pu

rgP

la2

g6

Ge

min

5G

on

4l

C1

dL

np

ep

Usp

48

Ctd

sp

lV

sig

10

lD

tnb

p1

Ra

nb

p1

0S

dh

bF

ab

p5

Iqg

ap

3A

rsg

Pa

rlD

ctn

6U

cp

2E

lovl6

Pa

qr7

Cm

as

Do

nso

nN

um

a1

Plc

g2

1

10

100

1000

10000 MockHSV-1

FPKM

H

HSV-1

Mock

050100

050100

92350000 91360000 91370000

Cxcl10 Cxcl11

HSV-1

Mock

040800

4080

82340000 82360000 82380000

Il1582400000

ChIP-seq H3K27Ac

HSV-1

Mock

075150

075150

53765000 53775000 53785000

Irf1

HSV-1

Mock

075150

075150

114910000 114930000 114950000

Oasl1

HSV-1

Mock

04080

04080

167300000 167320000 167340000

Tlr7

I

Figure S7. Super-enhancers were activated by viral infection. Related to Figure 7.(A-D) Heatmaps showing super-enhancers

activated (A) or repressed (B) with VSV infection and super-enhancers activated (C) or repressed (D) with HSV-1 infection. (E) Heatmap

showing activated super-enhancers with VSV and HSV-1 infection; histogram showing the expression level of genes according to the

appeared super-enhancers based on RNA-seq data in BMDMs infected with HSV-1 for 6 hours, and in wild-type or Safa–/– BMDMs

infected with HSV-1 for 6 hours (bottom). (F) Histogram of the combined score for Reactome biological processes for the activated and

repressed super-enhancers related genes after virus infection. (G) WT and Safa–/– iBMDMs were infected with VSV, and harvested for

ChIP with H3K27Ac antibody.(H) Heatmap showing repressed super-enhancers with VSV and HSV-1 infection ; histogram showing the

expression level of genes according to the repressed super-enhancers based on RNA-seq data in BMDMs infected with HSV-1 for 6hours (bottom). (I) Histogram (buttom) of ChIP-seq signal based on H3K27Ac occupancy.

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

RN

A-s

eq

(FK

PM

)C

hIP

-se

q(F

PK

M)

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

** * ** **

***

****

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT

-HS

V-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

*

*

**

**

***

**

****

Tlr13

B

WT

WT-H

SV

-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT-H

SV

-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT-H

SV

-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT-H

SV

-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

WT

WT-H

SV

-1

Sa

fa–

/–-H

SV

-1

Sa

fa–

/–

AC

The expression of NFκB targeted genes

E

WT

Sa

fa–/–

WT

－ － ＋ ＋

Sa

fa–/–

11

.9 7

.99

5.3

9 0

.17

-6.3

5

HSV-1

Down-regulation

Up-regulationNo-change

9

1346

Cytokines

2

814

Transcription

factors

14 12

Receptors

19 20

Adaptors

914

Kinases

WT

WT

－ － ＋ ＋

WT

WT

－ － ＋ ＋

WT

WT

－ － ＋ ＋

WT

WT

－ － ＋ ＋

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

Sa

fa–/–

F

D

Figure S8. SAFA associated super-enhancers potentiate antiviral responses. Related to Figure 7. (A) ChIP-qRT-PCR experiments

showing the super-enhancer activity of genes was decreased in Safa–/– iBMDMs (upper); RNA-seq showing HSV-1 induced genes

production was impaired in Safa–/– iBMDMs (bottom). (B) Histogram showing H3K27Ac ChIP-seq signal of indicated super-enhancers

with VSV and HSV-1 infection (upper), histogram showing the expression level of genes accordingly based on RNA-seq data in BMDMsinfected with HSV-1 for 6 hours (bottom). (C) The expression level of NFκB targeted genes in wild-type and Safa–/– BMDMs with HSV-1

for 6 hours. (D) Normalized profile of H3K27Ac ChIP-seq signal of indicated antiviral genes. (E) SAFA deficiency affected different gene

expression upon virus infection, including receptors, adaptors, kinases, transcription factors and cytokines. BMDMs derived from wild-

type or Safa–/– mice were infected with HSV-1 for 6 h followed by RNA-seq. (F) ChIP-qRT-PCR experiments showing SAFA binding to

IFNB enhancers depend on SPRY domain. SAFA–/– Hela cells were transfected with indicated plasmids, then infected with HSV-1 for 8hours, and cells were harvested for ChIP with Flag antibody.

─/─

A WTMAVS－/－

Mock

HS

V-1

Mo

ck

HS

V-1

HSV-1

IFNβ

GAPDH

- + - +WT MAVS-/-

D

B

IRF3

Tubulin

11

#

16

#

19

#

WT

IRF3－/－(crispr-cas9)

IRF3 －/－

SAFA

Tubulin

SAFACrispr - +

C

E

F

Tubulin

p-IRF3

Ve

c

SA

FA

MA

VS

Sting+

cG

AS

Trif

HA-Trif

Flag-cGAS

Flag-SAFA

Flag-Sting

Flag-MAVS

G

Tubulin

p-IRF3

Mo

ck

VS

V

HS

V-1

Poly

(I:

C)

WT

Mo

ck

VS

V

HS

V-1

Poly

(I:

C)

VS

V

Mo

ck

VS

V

Mo

ck

Nuclear Cytoplasm

WT Safa─/─

VS

V

Mo

ck

VS

V

Mo

ck

WT

IRF3

LaminB

Tubulin

Safa─/─

H

I J

K

0 h 3 h 9 h 1 2 h

0 .0 0 0 0

0 .0 0 0 2

0 .0 0 0 4

0 .0 0 0 6

0 .0 0 0 8

0 .0 0 6

0 .0 0 8

0 .0 1 0

Ifn

mR

NA

(re

lati

ve

) W T

S a fa- / -

L P S

N S

N S N S

L M

VSV

LP

S

LP

S

VSV ＋ ＋ － －

SAFA

－

LPS ＋ ＋－ －－

DSS － ＋ － ＋－

N

O P

Irf3─/─

Irf3─/─-Safa─/─

WT

Safa─/─

Safa─/─

Figure S9. SAFA facilitates extra-nuclear antiviral signaling. Related to Figure 7. (A) Wild-type and MAVS－/－ HEK293T cells were

infected with HSV-1. 6 hours later, IFNB1 mRNA was detected by regular PCR. (B) WT, Safa－/－, Tbk1－/－ and Safa－/－Tbk1－/－ MEF

cells were infected with HSV-1 or VSV. 8 hours later, the supernatants were transferred to L929-ISRE Luc cells, and 6 hours later

luciferase activity was measured.(C) Irf3–/– and Safa–/–Irf3–/– iBMDMs were infected with VSV. 24 hours later, type I IFNs were measured

by bioassay. VSV viral load was analyzed by plaque assay. (D) Immunoblotting showing IRF3 protein level in different Irf3－/－ iBMDM cell

monoclones and WT iBMDM cells. We used 19# monoclone in this paper. (E) Immunoblotting showing SAFA protein level in Irf3－/－ and

Safa－/－Irf3－/－ iBMDM cells. (F) HeLa cells were transfected with empty vector (Vec) or indicated plasmids, and 24 hours later indicated

protein levels were detected by immunoblotting. (G) Peritoneal macrophages derived from Safafl/fl (WT) and Safafl/fl -Lyz2 Cre (Safa–/–)

mice were infected with HSV-1, VSV for 6 hours, or with poly(I:C), LPS treatment for 4 hours, and indicated protein levels were detected

by immunoblotting. (H) Wild-type and Safa－/－ iBMDM cells were infected with VSV for 6 hours, proteins were extracted for nuclear-

cytoplasmic fractionation and resolved by immunoblotting. (I) Wild-type, Mavs–/–, Sting–/–, Tbk1–/–, Irf3–/– and Elf4–/– iMEF cells were

transfected with IFNβ–Luc plasmid with empty vector or SAFA plasmid, 24 hours later infected with VSV, and 24 hours later luciferase

activity was measured. (J) Wild-type and SAFA–/– iMEF cells were transfected with empty vector, MAVS, STING or TBK1 plasmid, 24

hours later mRNA was extracted and Ifnb1 mRNA was detected by qRT-PCR. (K) iMEF cells were transfected with indicated reporter

plasmid with empty vector, MAVS, STING, TBK1, IRF3 or ELF4 plasmid, 24 hours later luciferase activity was measured. (L)Peritoneal

macrophages derived from Safafl/fl (WT) and Safafl/fl -Lyz2 Cre (Safa–/–) mice were infected with HSV-1, VSV for 6 hours, or with poly(I:C),

LPS treatment for 4 hours, and Ifnb1 mRNA were measured by qRT-PCR. (M) Peritoneal macrophages derived from Safafl/fl (WT) and

Safafl/fl -Lyz2 Cre (Safa–/–) mice were infected with VSV or treated with LPS for indicated times, and Ifnb1 mRNA were measured by qRT-

PCR. (N) Peritoneal macrophages were infected with VSV for 6 hours or treated with LPS for 4 hours and followed by Oligomerization

assay. (O) Wild type and distal IFNβ enhancer mutant macrophages were treated with LPS for 4 hours, and Ifnb1 mRNA were measured

by qRT-PCR. (P) The luciferase activity of indicated reports was measure after transfection with SAFA and infection with VSV.