TRIM28/KAP1 promotes cell proliferation, tumor growth and metastasis of breast cancer cells and regulates the

expression of multiple KRAB-ZNFs

Joseph B. Addison, Colton Koontz, James H. Fugett, Chad J. Creighton, Dongquan Chen, Mark K. Farrugia, Renata R. Padon, Maria A. Voronkova, Sarah L. McLaughlin, Ryan H.

Livengood, Chen-Chung Lin, J. Michael Ruppert, Elena N. Pugacheva, and Alexey V. Ivanov

Supplementary Figures

ZNF10: Linker ZnF-2-1:-NIHLIQFARTHTGDKSYKCPDNDNSLTH- ZnF-1-1: -GSSLGISKGIHREKPYECKECGKFFSW- ZnF1-2: -RSNLTRHQLIHTGEKPYECKECGKSFSR- ZnF2-3: -SSHLIGHQKTHTGEEPYECKECGKSFSW- ZnF3-4: -FSHLVTHQRTHTGDKLYTCNQCGKSFVH– ZnF4-5: -SSRLIRHQRTHTGEKPYECPECGKSFRQ- ZnF5-6: -STHLILHQRTHVRVRPYECNECGKSYSQ- ZnF6-7: -RSHLVVHHRIHTGLKPFECKDCGKCFSR- ZnF7-8: -SSHLYSHQRTHTGEKPYECHDCGKSFSQ- ZnF8-9: -SSALIVHQRIHTGEKPYECCQCGKAFIR- ZnF9-10:-KNDLIKHQRIHVGEETYKCNQCGIIFSQ- ZnF10-1:-NSPFIVHQIAHTGEQFLTCNQCGTALVN-

ZNF263: Linker ZnF1-2: -NTHLTRHQRTHTGEKPYQCNICGKCFSC- ZnF2-3: -NSNLHRHQRTHTGEKPYKCPECGEIFAH- ZnF3-4: -SSNLLRHQRIHTGERPYKCPECGKSFSR- ZnF4-5: -SSHLVIHERTHERERLYPFSECGEAVSD- ZnF5-6: -GMHLTRHQRTHTGEKPYKCTLCGENFSH- ZnF6-7: -RSNLIRHQRIHTGEKPYTCHECGDSFSH- ZnF7-8: -SSNRIRHLRTHTGERPYKCSECGESFSR-

ZNF350: Linker ZnF1-2: -KSWLTDHQVMHTGEKPHRCSLCEKAFS- ZnF2-3: -KFMLTEHQRTHTGEKPYECPECGKAFL- ZnF3-4: -KSRLNIHQKTHTGEKPYICSECGKGFI- ZnF4-5: -KGNLIVHQRIHTGEKPYICNECGKGFI- ZnF5-6: -KTCLIAHQRFHTGKTPFVCSECGKSCS- ZnF6-7: -KSGLIKHQRIHTGEKPFECSECGKAFS- ZnF7-8: -KQKLIVHQRTHTGERPYGCNECGKAFA-

Figure S1. KRAB-ZNF proteins contain multiple copies of the zinc finger linker (ZnFL) motif. Alignment of linker sequences between zinc fingers from three randomly selected human KRAB-ZNF proteins. Each line contains two histidines (H) from the preceding zinc finger and two cysteines (C) from the following zinc finger shaded in grey. Highly conserved zinc finger linker TGEKPY residues are shaded in yellow.

Mouse

Drosophila

Zebrafish

Xenopus

Chicken

Human

Figure S2. Zinc finger linker conservation in different species. Logo representation of amino acid conservation in 28-aa zinc finger domains of C2H2-type ZNF proteins from different species. A high bit score reflects invariant residues C, C, H and H. 7-aa zinc finger linker sequence TGEKPYK/E is highly conserved. Red arrows show prevalence of R and F in chicken proteins.

Human cancer cell lines

Ubc9

KAP1

a-ZnFL

Be

as

2B

A5

49

H4

60

H1

39

5H

12

99

HM

LE

ZR

75

-1M

DA

46

8

H3

58

NTe

ra2

Ju

rka

tK

56

2H

ep

G2

Lung Breast Em

bry

o

Blo

od

Liv

er

1 2 3 4 5 6 7 8 9 10 11 12 13

11580

kDa

65

50

30

25

KAP1*SUMO115

15

Figure S3. Ubiquitous expression of KAP1 and ZNFs in human cancer cells lines. Western blot analysis of KAP1 and ZNFs in different human cancer cells lines of lung, breast, embryo, blood and liver origin. Ubc9 serves as loading control.

_________________________________________________________________Dataset (# samples) Fold up TRIM28/KAP1 P-value (tumor vs. normal)_________________________________________________________________Radvanyi Breast (35 vs 9 normal)

Invasive Ductal BC (32) 1.648 0.016Ductal BC in Situ (3) 1.883 0.046

Sorlie Breast 2 (93 vs 4 normal)Ductal BC (93) 1.397 0.032

Gluck Breast (154 vs 4 normal)Invasive BC (154) 1.270 0.014 

TCGA Breast array (518 vs 61 normal)Mucinous BC (4) 2.062 0.002Invasive BC (76) 1.540 6.76E-10Invasive Ductal & Lobular BC (3) 1.305 0.018Invasive Lobular BC (36) 1.339 1.41E-5Invasive Ductal BC (392) 1.439 2.50E-9Mixed Lobular & Ductal BC (7) 1.247 0.024

_________________________________________________________________

Figure S4. KAP1 is overexpressed in human breast cancer. Microarray analyses of Trim28/KAP1 expression in four different breast datasets from Oncomine.com.

Trim28/KAP1

-1.5

-1

-0.5

0

0.5

1

1.5

2

[1] Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, Hu Z, et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome biology. 2007;8:R76

Normal Mesench. Basal Luminal Mixed

Figure S5. KAP1 is overexpressed in mouse mammary tumors. Microarray analysis of Trim28/KAP1 expression in 108 tumors from 13 different mouse models of breast cancer. Normalized expression data were downloaded from [1] and plotted for Trim28/KAP1 on log2-transformed scale. Group assignments are from [1]. Group I represents normal mammary tissue.

Figure S6. KRAB-ZNFs and non-KRAB-ZNFs are overexpressed in breast tumors. Expression analysis of 343 KRAB-ZNF and 370 non-KRAB-ZNF genes in TCGA RNA-Seq (n=896) dataset from Supplementary File 1. For all genes P < 0.05, two-tailed t test.

ZNFs tumor/normal

Num

ber

of g

enes

KRAB (343) non-KRAB (370)0

50

100

Up >1.35-foldUp >2-foldDown >1.35-foldDown >2-fold

N T N T N T N T N T T N N T N T N T N T N T N T N T T T

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15: matched

Breast tissue biopsy samples

KAP1

non-spec.

N – normal, T – tumor

pairs

Stage: IIIC IIA I IIB IIA IIIC IIIB IIA IIA IIIB IIA IIA IIIA IV

kDa

11580

65

50

Figure S7. KAP1 protein is overexpressed in human breast tumors. Western blot analysis of KAP1 protein expression in biopsy samples of matched normal (N)–tumor (T) pairs obtained from the same patients. Stages of breast cancers are shown above. Non-specific band serves as loading control.

Figure S8. KAP1 SUMOylation level is increased in breast cancer cell lines. Percentage of SUMOylated KAP1 relative to total KAP1 protein level in each cell line based on densitometric quantification of protein bands from Fig. 3B. Data shown as mean ±SD. ns – non-significant, * - P < 0.05, ** - P < 0.01, two-tailed t test.

% S

UM

Oyl

ate

d K

AP

1

HM

EC

1H

ME

C2

HM

LE

MC

F1

0A

HC

C1

50

0Z

R7

5-1

MD

A-M

B-4

53

MD

A-M

B-3

61

HC

C1

42

8M

CF

7H

CC

11

43

HC

C1

93

7B

T2

0M

DA

-MB

-46

8M

DA

-MB

-15

7H

CC

13

95

HC

C3

8M

DA

-MB

-23

1L

NM

DA

-MB

-23

1B

T5

49

HS

57

8T

0

10

20

30

40

ns ns

ns

ns n

s*

**

*

**

**

* * * ***

**

**

* *

1 2 3 4

KAP1

RIP

A

RIP

A+

NE

M

mIP

La

em

mli

80

kDa

115

Tubulin

GAPDH

KAP1*SUMO

: buffers

mIP – no SDS (0.5% NP-40, 0.5% Triton X-100)RIPA – 0.1% SDSLaemmli – 2% SDS

30

50

Figure S9. KAP1 SUMO modification is preserved by N-ethylmaleimide (NEM) and SDS . Western blot analysis of KAP1 in MDA-MB-453 cells lysed in different buffer conditions (mIP – no SDS, RIPA – 0.1% SDS, Laemmli – 2% SDS). An inhibitor of de-SUMOylating enzymes, NEM was added to lysis buffer to final concentration 10mM. GAPDH and Tubulin serve as loading controls.

Knockdown (KD) Overexpression (OX)

Figure S10. Extent of KAP1 knockdown and overexpression. RT-qPCR analysis of KAP1 mRNA in the indicated cell lines from Fig.4A and Fig.6. KAP1 mRNA levels for controls were assigned the relative value of 1. Data shown as mean ±SEM. In all cell lines KD and OX compared to control P < 0.001, two-tailed t test.

HMLE

ZR75

MDA-M

B-468

MDA-M

B-231

LN

0.00

0.05

0.10

0.15

0.200.8

1.0

1.2shControlshKAP1-3

Rel

ativ

e fo

ld c

han

ge

shKAP1-4

HMLE

ZR75

MDA-M

B-468

MDA-M

B-231

LN

0

10

20

30VectorKAP1-WTKAP1-M2

Rel

ativ

e fo

ld c

han

ge

Schematic representation of KAP1 functional mutants

Figure S11. Schematic representation of KAP1 functional mutants. A model for sumoylation-dependent, KAP1-mediated gene silencing. The SUMO-conjugated KAP1 recruits the NuRD complex and SETDB1 through SUMO interactions, which results in the deacetylation of histones and the methylation of histone H3-K9. KAP1-bound HP1 recognizes H3-K9 methylation. Point mutations (in red) were introduced in KAP1 at three separate regions which hinder specific KAP1 interactions. KAP1-R, KAP1-B2 and KAP1-C2 mutants are deficient in KRAB domain binding, KAP1-M2 mutant is deficient in interaction with HP1 and KAP1-K6R mutant is deficient in SUMOylation. Small triangle, acetyl mark; small circle, H3-K9 trimethyl mark on histone tails.

SETDB1

DNA Binding Site

Zn Zn

ZnZnZnZnZn

ZnZn

Histone deacetylation, H3-K9 methylationand HP1 deposition

SUMO

SUMO CHD3

KRABKAP1

NuRD

HDAC1/2

HP1 Transcription OFF

Tumor Suppressor Geneor Oncogene

SETDB1

DNA Binding Site

Zn Zn

ZnZnZnZnZnZn Zn

ZnZnZnZnZnZnZnZn

ZnZn

Histone deacetylation, H3-K9 methylationand HP1 deposition

SUMOSUMO

SUMOSUMO CHD3

KRABKAP1

NuRD

HDAC1/2

HP1 Transcription OFF

Tumor Suppressor Geneor Oncogene

C2M2 K6R

KAP1 functional point mutants

RB2

MEF Kap1 -/-

doseKAP1

a-ZnFL

KAP1

kDa115

80

65

50

3025

115

30

Vect

orK

AP

1-W

T

KA

P1-

R

KA

P1-

B2

KA

P1-

M2-

K6R

GAPDH

KA

P1-

C2

Figure S12. Expression of KRAB-ZNF proteins depends on their ability to interact with KAP1 . WB analysis of KAP1 and KRAB-ZNFs in Kap1 -/- MEFs transfected with vector, wild type KAP1 and the indicated KAP1 mutants. Increasing doses of KAP1 expression constructs were used as shown. GAPDH serves as loading controls. WT – wild type KAP1. R, B2 and C2 are three KAP1 RBCC domain mutants defective in interaction with the KRAB domain. R mutant - RING domain mutation CC65,68AA; B2 mutant - Box2 domain mutation C209A,H212A and C2 mutant - coiled-coil mutation L306P (ref. 40). M2-K6R double mutant is defective in HP1 interaction and SUMOylation (both required for transcriptional repression) but capable of binding to the KRAB domain.

Fig. S13. KAP1 knockdown in MDA-MB-231LN cells inhibits primary tumor growth in orthotopic xenograft mouse model. Tumor growth curves of orthotopically injected MDA-MB-231LN cells: shControl (n=6), shKAP1-3 (n=5), shKAP1-4 (n=5). Data shown as mean + SEM, * - P < 0.05, ** - P < 0.01, two-tailed Student’s t test.

Weeks

Tu

mo

r vo

lum

e, m

m3

2 3 4 5 60

1000

2000

3000

shControlshKAP1-3shKAP1-4

*

*

**