Science - USA (2021-10-29)

Rb appears to enhance the transcriptional
activity of these TFs in SNCs to establish
the PASP.
Analysis of publicly available Rb ChIP-seq
data from OI-senescent IMR-90 cells ( 25 ) re-
vealed that Rb peaks mapped to the promoter
regions of 948 SFs and that these peaks were
enriched for binding sites of all TFs that we
identified as instrumental in establishing the
PASP, with the exception of RELA (Fig. 1D
and table S5). Rb peaks mapped to promoter
regions of 49 of 172 PASP genes identified in
IR IMR-90 cells and associated with TFs critical
for establishing the PASP (Fig. 1E and table S5).
Most of these promoter regions had no such
peaks when IMR-90 cells were cycling or qui-
escent. Furthermore, SMAD2, SMAD3, STAT1,
and STAT6 coimmunoprecipitated Rb from IR-
senescent MEFs, and codepletion of SMAD2,
SMAD3, STAT1, and STAT6 in IR-senescent
MEFs reduced transcription of PASP genes

where Rb and these TFs colocalize in pro-

moter regions (fig. S6). Thus, a p21-responsive

Rb pool interacts with specific SMAD and

STAT TFs at PASP gene promoters to enhance

their expression.

p21 simultaneously places cells under

proliferative arrest and immunosurveillance

To determine whether the PASP is senescence

dependent, we performed RNA-seq on non-

senescentMEFswithhighp21collected2or

4 days (d2 or d4) after irradiation (Fig. 2A and

fig.S7,AtoD).Atd2andd4,IRMEFsup-

regulated 351 and 450 SFs, respectively, 241 of

which were shared with d10 IR MEFs (Fig. 2A

and table S6). At d4, IR MEFs depleted forp21

orRb, lost 235 and 171 of their SFs, respec-

tively, indicating that the PASP is a senescence-

independent phenomenon (Fig. 2B and fig. S7,

A to D). Eighty-four PASP factors were com-

monly lost in d4 and d10 IR MEFs when p21

or Rb was depleted, demonstrating that the

PASP of non-SNCs becomes an integral part

of the SASP as cells advance to a senescent

state(Fig.2Bandfig.S7,EandF).

Functional annotation analysis on the 84

shared PASP factors indicated that several

traits of SNCs may be p21-Rb dependent, in-

cluding features involving cell migration and

adhesion and the immune system (Fig. 2C and

fig. S7G). This raises the possibility that the

PASP plays a role in immunosurveillance. To

test this idea, we determined the extent to

which the PASP affects the migratory behavior

of mouse peritoneal immune cells in a trans-

well system (Fig. 2D). Conditioned medium

from d4 nonsenescent (CM-NS) or d10 senes-

cent (CM-S) IR MEFs promoted transwell mi-

gration of macrophages, a property that was

lost with CM-NS and CM-S fromp21-orRb-

depleted IR MEFs (Fig. 2E). None of the CMs

affected lymphocyte migration in this assay

Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 2 of 15

Adam15

IRIR:

shp21

IR:

shRb

97 70 36

shp21

vs shScr

(167)

shRb

vs shScr

(106)

C

IGFBP3

SERPINE1

GDF11

VEGFA

ANGPTL4

IR IMR-90

SASP factors

A

Gas6

Cxcl14

Psap

Igfbp3

Mmp19

shp21

vs shScr

(188)

shRb

vs shScr

(161) IRIR:

shp21

IR:

shRb

IR MEFs

SASP factors

54 134 27

B

+1 –1

Log 2 FC

IRIR:

shp21

IR:

shRb

RELA

NFB1

REL

CEBP

SMAD2

SMAD3

STAT1

STAT3

STAT5A

STAT5B

STAT6

IRRE

P

OIIR:

shp21

IR:

shRb

10 1.3

–Log 10 (FDR)

1.3 10

RELA

NFB1

CEBP

SMAD2

SMAD3

STAT1

STAT5A

STAT5B

STAT6

IR MEFs IR IMR-90 MEFs IMR-90

D

E

ADAM15

Cycling IMR-90

OI IMR-90

Quiescent IMR-90

Rb peak with

STAT1/3 & SMAD2/3 motifs

ChIP-Seq 10,275

genes with

Rb peaks

TF motif

enrichment

analysis

OI IMR-90 Rb

FN1

Rb peaks with

SMAD2/3 motifs

PSAP

Rb peak with

SMAD2/3 motifs

GAS6

Rb peaks with

STAT1/3 & SMAD2/3 motifs

948 genes

encoding

secreted

factors

SMAD2

SMAD3

STAT1

STAT3

STAT6

432

627

225

358

353

1.94 e-9

4.77 e-36

2.32 e-15

2.27 e-16

5.1 e-2

padj

Genes with

TF motif TF motif

Fn1

ADAM15

Fig. 1. p21-activated Rb interacts with STAT and SMAD TFs at select gene
promoters to establish a bioactive secretome.(A) Venn diagrams of RNA-seq
data depicting down-regulated SASP factors with depletion ofp21orRbin the
indicated SNCs. (B) Heatmaps of commonly down-regulated SASP factors
indicated in (A). FC, fold change. (C) Identification of TFs that transcriptionally
activate genes encoding PASP factors using overrepresentation analyses on

RNA-seq data from IR-, REP, OI-senescent MEFs, IR-senescent IMR-90 cells, and

their nonsenescent counterparts. Bolded TFs are significantly activated in SNCs

and inhibited upon shRNA-mediated depletion ofp21orRb. FDR, false discovery rate.

(D) Identification of SASP genes that bind Rb, and TF motif analysis of Rb peaks

underlying SFs in OI-senescent IMR-90 cells. (E) Representative Rb occupancy plots

at PASP genes. Experiments in the above panels were performed once.

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