phospho-STAT5

total-STAT5

β-actin

β-actin

DIP mixFull medium

DMSO300nM TSA - + - +

+ - + -+ + - -- - + +

Histone H3

TRPS1

Cytoplasmic

HDAC1

CHD4

GAPDH

HDAC2

RBBP7

TOP1

Membranous Soluble-nuclear Chromatin-bound

sgTr

ps1

#1 +

EV

sgTr

ps1

#1 +

TRP

S1tru

nc

sgSc

r #1

+ EV

sgTr

ps1

#1 +

EV

sgTr

ps1

#1 +

TRP

S1tru

nc

sgSc

r #1

+ EV

sgTr

ps1

#1 +

EV

sgTr

ps1

#1 +

TRP

S1tru

nc

sgSc

r #1

+ EV

sgTr

ps1

#1 +

EV

sgTr

ps1

#1 +

TRP

S1tru

nc

sgSc

r #1

+ EV

FL

TR

B

C

F G

2 3 4

887aa686aa309aaZinc fingerNLS

0.0

0.5

1.0

Full mediumDIP mixTSA (nM)

---+++

300800

Rel

ativ

e β-

case

in e

xpre

ssio

n(n

orm

aliz

ed to

β-a

ctin

)

-+0

***

1 2 3 4 5 6

GATATGGCCTGCACCCCATCAGG CCTCCGAGGGTGTGAAACCTTCAClones #1 + #3Clone #2

A Cornelissen_FigS2

HDAC1

β-actin

TRPS1trunc - + - +EV + - + -

sgScr #1 sgTrps1 #1

β-actin

HDAC2

TRPS1FL

TR

TRPS1trunc - + - +EV + - + -

sgScr #1 sgTrps1 #1D

E

0 50 100 150 2000

50

100

sgScr #1 + EVsgScr #1 + TRPS1trunc

sgTrps1 #1 + EVsgTrps1 #1 + TRPS1trunc

Time (hours)

Con

fluen

cy (%

)

TRPS1

β-actin

shNT shTrps1 #2

0 20 40 60 80 1000

20

40

60

80

Time (hours)

shNT

shTrps1 #2

Con

fluen

cy (%

)

0.0

0.5

1.0

1.5

shNT

shTrps1 #2

DIP mix

-+-++-+-

++--

Rel

ativ

e β-

case

in e

xpre

ssio

n(n

orm

aliz

ed to

β-a

ctin

)

**

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e β-

case

in e

xpre

ssio

n(n

orm

aliz

ed to

β-a

ctin

)

EVTRPS1trunc

DIP mix

-+

+sgTrps1 #1

+-

+

-+

+sgScr #1

+-

+

+-

-sgScr

#1

+-

-sgTrps1

#1

ns

ns

H

I

J K

LsgScr #1 sgTrps1 #1

EVTRPS1trunc - +

+ -- ++ -

TRPS1

β-actin

FL

TR

phospho-STAT5

β-actin

#1 #2 #1 #2 #3

sgScr sgTrps1

total-STAT5

β-actin

TRPS1

#1 #2 #1 #3

sgScr sgTrps1

DIP mix + + + + + - - - -

M N

0

2

4

6

8

10

Maml Mkl1 Tcf3 Hoxd9 Klf3 Gata3(neg region)

HDAC2 ChIP

sgScr #1 + mTRPS1truncsgScr #1 + EV

sgTrps1 #1 + mTRPS1truncsgTrps1 #1 + EV

Fold

enr

ichm

ent

ns***

ns

ns**

ns

ns***

ns

ns***

ns

ns***

nsns

ns

ns

0

5

10

15

20

Fold

enr

ichm

ent

Maml Mkl1 Tcf3 Hoxd9 Klf3 Gata3(neg region)

HDAC1 ChIP

sgScr #1 + mTRPS1truncsgScr #1 + EV

sgTrps1 #1 + mTRPS1truncsgTrps1 #1 + EV

ns***

ns

ns*

ns

ns***

nsns

ns

ns

ns***

ns

ns

ns

ns

3

Supplementary Figure S2. Loss of TRPS1 affects HDAC1 and HDAC2 DNA binding and lactogenic

differentiation, while TRPS1trunc expression is dispensable. (A) Schematic representation of the Trps1

gene locus with the locations of the guide RNAs used to target Trps1 indicated. PAM sequences are

indicated in red. Numbered boxes represent exons of the canonical gene transcript. (B) Schematic

representation of TRPS1trunc cDNA reflecting loss of exon 5 and 6. Numbered boxes represent exons of

the canonical gene transcript. (C) Subcellular fractionation of HC11 cells with expression full-length

TRPS1, TRPS1 deficient or expression of TRPS1trunc. NuRD complex members are indicated with a green

lining. GAPDH was used as a marker for the cytoplasmic fraction, Topoisomerase 1 (TOP1) for the

soluble-nuclear fraction and histone H3 for the chromatin-bound fraction. Black arrowhead, full-length

TRPS1 (FL); grey arrowhead, truncated TRPS1 (TR). (D) Western blot analysis of HDAC1 and HDAC2

expression in HC11 control and TRPS1 knockout cells transduced with a control or TRPS1trunc construct.

β-actin was used as a loading control. Black arrowhead, full-length TRPS1 (FL); grey arrowhead,

truncated TRPS1 (TR). (E) HDAC1 and HDAC2 ChIP-qPCR analysis in HC11 control and TRPS1

knockout cells transduced with a control or TRPS1trunc construct. Five target regions and one negative

control region are shown. Data represent mean+standard deviation (SD). Two-way ANOVA: *** p<0.001,

** p<0.01, * p<0.05, ns p>0.05. (F) Western blot analysis of phospho-STAT5 and total-STAT5 expression

in HC11 cells treated with DIP mix compared to full medium, in combination with DMSO or 300nM

Trichostatin A (TSA). (G) RT-qPCR analysis for β-casein (Csn2) expression in HC11 cells treated with

DIP mix, in combination with DMSO, 80nM or 300nM TSA. Data represent mean+SD, n=2. One-way

ANOVA: ** p<0.01, * p<0.05. (H) Western blot analysis of phospho-STAT5 and total-STAT5 expression in

HC11 control and TRPS1 knockout cells treated with or without DIP mix. β-actin was used as a loading

control. (I) Western blot analysis of TRPS1 expression in HC11 cells transduced with a non-targeting

shRNA or shRNA #2 targeting Trps1 (as indicated in Figure S1B). β-actin was used as a loading control.

(J) Cell proliferation of HC11 cells transduced with a non-targeting shRNA or an shRNA targeting Trps1,

as quantified using IncuCyte imaging for 100 hours. Data represent mean±SEM. (K) RT-qPCR analysis of

β-casein (Csn2) expression levels in HC11 cells transduced with a non-targeting shRNA or targeting

Trps1 upon treatment with DIP mix. Data represent mean+SD, n=3. T-test: ** p<0.01. (L) Western blot

analysis of TRPS1 expression in HC11 control and TRPS1 knockout cells transduced with a control or

4

TRPS1trunc construct. β-actin was used as a loading control. Black arrowhead, full-length TRPS1 (FL);

grey arrowhead, truncated TRPS1 (TR). (M) Cell proliferation of HC11 control and TRPS1 knockout cells

transduced with a control or TRPS1trunc construct, as quantified using IncuCyte imaging for 200 hours.

Data represent mean±SEM. (N) RT-qPCR analysis of β-casein (Csn2) expression in HC11 control and

TRPS1 knockout cells transduced with a control or TRPS1trunc construct upon treatment with DIP mix.

Data represent mean+SD, n=2. One-way ANOVA: ns p>0.05.