Supplementary Information

MicroRNA-33b knock-in mice for an intron of sterol regulatory

element-binding factor 1 (Srebf1) exhibit reduced HDL-C in vivo

Takahiro Horie, Tomohiro Nishino, Osamu Baba, Yasuhide Kuwabara, Tetsushi Nakao,

Masataka Nishiga, Shunsuke Usami, Masayasu Izuhara, Fumiko Nakazeki, Yuya Ide, Satoshi

Koyama, Naoya Sowa, Naoya Yahagi, Hitoshi Shimano, Tomoyuki Nakamura, Koji Hasegawa,

Noriaki Kume, Masayuki Yokode, Toru Kita, Takeshi Kimura, and Koh Ono

Supplementary Table S1

Serum lipid profiling of WT and miR-33b KI+/+ mice by HPLC

Lipoprotein

Major (Fraction No.)

Diameter Sub-class (Fraction No.) WT (n=4) KI+/+ (n=5)

TC (mg/dl) 72.71 ± 5.42 52.56 ± 3.91*

CM (1-2) >80nm 0.73 ± 0.14 0.36 ± 0.06*

VLDL (3-7) 30-80nm 3.93 ± 0.22 3.76 ± 0.41

LDL (8-13) 16-30nm 10.86 ± 0.60 9.85 ± 0.78

large LDL (8) 3.10 ± 0.09 3.39 ± 0.24

medium LDL (9) 2.85 ± 0.11 3.15 ± 0.21

small LDL (10) 1.77 ± 0.14 1.63 ± 0.18

very small LDL (11-13) 2.73 ± 0.60 1.68 ± 0.26

HDL (14-20) 8-16nm 57.20 ± 4.92 38.59 ± 3.23*

very large HDL (14-15) 2.30 ± 0.50 0.68 ± 0.14*

large HDL (16) 18.49 ± 2.44 8.89 ± 1.38**

medium HDL (17) 24.90 ± 1.46 18.97 ± 1.29*

small HDL (18) 8.45 ± 0.43 7.74 ± 0.47

very small HDL (19-20) 3.05 ± 0.25 2.32 ± 0.25

TG (mg/dl) 22.04 ± 2.48 19.27 ± 2.66

Values are mean ± SEM. Blood was obtained from chow-fed 8 weeks old male mice, after 4 h fasting.

The serum was analyzed by HPLC, as described in material and methods. *p<0.05, **p<0.01 compared with WT mice.

TC, total cholesterol; TG, triglyceride; CM, chylomicrons; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein;

HDL, high-density lipoprotein.

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Human SREBF1Mouse Srebf1

Exon 16

Exon 17

Exon 18

1 GAGAGACCCCTGCCCAGGGCAGCTCTGCACTCCTTCAAGGCTGCCCGGGCCCTGCTGGGC 601 GAGAGACCCCTGCCCAGGGCAGCTCTGTACTCCTTCAAGGCTGCCCGGGCTCTGCTGGAC 60

61 TGTGCCAAGGCAGAGTCTGGTCCAGCCAGCCTGACCATCTGTGAGAAGGCCAGTGGGTAC 12061 CACAGAAAGGTGGAATCTAGCCCAGCCAGCCTGGCCATCTGTGAGAAGGCCAGTGGGTAC 120

121 CTGCAGGACAGCCTGGCTACCACACCAGCCAGCAGCTCCATTGACAAGGCCGTGCAGCTG 180121 CTGCGGGACAGCTTAGCCTCTACACCAACTGGCAGTTCCATTGACAAGGCCATGCAGCTG 180

181 TTCCTGTGTGACCTGCTTCTTGTGGTGCGCACCAGCCTGTGGCGGCAGCAGCAGCCCCCG 240181 CTCCTGTGTGATCTACTTCTTGTGGCCCGTACCAGTCTGTGGCAGCGGCAGCAGTCACCA 240

241 GCCCCGGCCCCAGCAGCCCAGGGCACCAGCAGCAGGCCCCAGGCTTCCGCCCTTGAGCTG 300241 GCTTCAGTCCAGGTAGCTCACGGTACCAGCAATGGACCCCAGGCCTCTGCTCTGGAGCTG 300

301 CGTGGCTTCCAACGGGACCTGAGCAGCCTGAGGCGGCTGGCACAGAGCTTCCGGCCCGCC 360301 CGTGGTTTCCAACATGACCTGAGCAGCCTGCGGCGGTTGGCACAGAGCTTCCGGCCTGCT 360

361 ATGCGGAGG 369361 ATGAGGAGG 369

Srebf1 Srebf2Mouse

miR-33(a)SREBP-2SREBP-1

SREBF1 SREBF2Human

miR-33b miR-33aSREBP-2SREBP-1

miR-33a; GUGCAUUGUAGUUGCAUUGCAmiR-33b; GUGCAUUGCUGUUGCAUUGC

Ex.16 Ex.17miR-33b

166bp 201bp475bp

Ex.16 Ex.17

166bp 201bp410bp

Ex.15 Ex.18

133bp

HumanSREBF1

MouseSrebf1

204bp 112bp

Ex.18

112bp

Ex.15

133bp 168bp

89bp

98bp

a

b

Supplementary Figure S1. Scheme of SREBFs and miR-33s and homologies in exons 16 and 17 of

SREBF1 between human and mouse. (a) miR-33b is lacking in intron of mouse Srebf1. Mature sequences

of miR-33a and miR-33b are indicated in the inset. (b) There are high homologies in exons 16 and 17 between

human SREBF1 and mouse Srebf1 (82.6% of nucleotides and 79.7% of amino acids).

a

bSr

ebf1

(A.U

.)

WT KI+/- KI+/+0.0

0.5

1.0

1.5

2.0c d

Sreb

f2 (A

.U.)

WT KI+/- KI+/+0.0

0.5

1.0

1.5

5’ probe

WT

3’ probe

Neo probe 6.6 kb

35.2 kb (WT allele)19.6 kb (Targeted allele)

10.7 kb (WT allele)

4.8 kb (Targeted allele)

ES clone for injection

ES clones

e f

miR

-33a

(A.U

.)

WT KI+/- KI+/+0.0

0.5

1.0

1.5

2.0

2.5***

*

Sreb

f1 (A

.U.)

Brain

Heart

Liver

Kidney

Sub WAT

Epi WAT

Gastro

cnem

ius0.0

0.5

1.0

1.5

miR

-33b

(A.U

.)

Brain

Heart

Liver

Kidney

Sub WAT

Epi WAT

Gastro

cnem

ius0.0

0.5

1.0

1.5

Supplementary Figure S2. Generation of miR-33b knock-in mice. (a) Southern blotting analysis of genomic DNA

from ES cells that were successfully targeted by a KI vector. Representative images are shown. (b-d) Quantitative

real-time PCR analysis of miR-33a (b), Srebf1 (c) and Srebf2 (d) in the liver of 8-wk-old WT, KI+/- and KI+/+ male

mice (n=6). Values are mean ± s.e.m. *p < 0.05 ***p < 0.001 by one-way analysis of variance. (e, f) Quantitative

real-time PCR analysis of Srebf1 (e) and miR-33b (f) in 8-wk-old KI+/+ male mice (n=3). Values are mean ± s.e.m.

Sub WAT indicates subcutaneous white adipose tissue. Epi WAT indicates epididymal white adipose tissue.

a b c d

miR

-33a

(A.U

.)

WT KI+/+0.0

0.5

1.0

1.5

miR

-33b

(A.U

.)

WT KI+/+0.0

0.5

1.0

1.5

N.D.

Sreb

f2 (A

.U.)

WT KI+/+0.0

0.5

1.0

1.5

Sreb

f1 (A

.U.)

WT KI+/+0.0

0.5

1.0

1.5

e f

ABC

A1 (%

)

WT KI+/+0

50

100

150

ABC

G1

(%)

WT KI+/+0

50

100

150

* *

Supplementary Figure S3. The expression levels of miR-33s and Srebfs in KI macrophages. (a-d)

Quantitative real-time PCR analysis of miR-33b (a), miR-33a (b), Srebf1 (c) and Srebf2 (d) in peritoneal

macrophages from 8-wk-old WT and KI+/+ male mice (n=3). Values are mean ± s.e.m. N.D., not determined. (e,

f) Densitomeric analysis of ABCA1 (e) and ABCG1 (f) in peritoneal macrophages from 8-wk-old WT and KI+/+

male mice. (n = 4). Values are mean ± s.e.m. *p < 0.05 by Student’s t-test.

a b c**

Live

r w

eigh

t (g)

Vehicl

e

T0901

317

0.0

0.5

1.0

1.5

2.0

**Li

ver

wei

ght (

g)

Vehicl

e

T0901

317

0.0

0.5

1.0

1.5

2.0

Supplementary Figure S4. Liver weight and Srebf1 expression of T0901317-treated mice. (a) Liver weight

of vehicle or T0901317-treated 8-wk-old KI+/+ male mice. Values are the means ± s.e.m. (n = 6, **p<0.01 by

Student’s t-test). (b) Liver weight of vehicle or T0901317-treated 8-wk-old WT male mice (n=4, **p<0.01 by

Student’s t-test). (c) Quantitative real-time PCR analysis of Srebf1 in the liver of vehicle or T0901317-treated

8-wk-old WT male mice (n=4, **p< 0.01 by Student’s t-test).

**

Sreb

f1 (A

.U.)

Vehicl

e

T0901

317

0

1

2

3

a b cAB

CA1

(%)

WT KI+/- KI+/+0

50

100

150

d e fM

atur

e SR

EBP-

1 (%

)

WT KI+/- KI+/+0

50

100

150

SRC

1 (%

)

WT KI+/- KI+/+0

50

100

150

200PC

K1

(%)

WT KI+/- KI+/+0

50

100

150

200C

REB

(%)

WT KI+/- KI+/+0

50

100

150

G6P

ase

(%)

WT KI+/- KI+/+0

50

100

150

Supplementary Figure S5. The expression levels of cholesterol and glucose metabolic genes. The protein

expression levels of ABCA1 (a), SREBP-1 (b), SRC1 (c), PCK1 (d), CREB (e) and G6Pase (f) in the liver of

8-wk-old WT, KI+/- and KI+/+ male mice by densitometry. The expression levels were normalized with β-actin or

TF2B expressions. The expression levels of WT mice were set at 100%. Values are mean ± s.e.m. *p < 0.05,

**p<0.01 ***p < 0.001 compared with WT by one-way analysis of variance (a-d; n=6, e and f; n=3).

*** *

*

**

SRE SREBP-1 responsive gene

SREBF1c miR-33b

miR-33b

InsulinLXR ligand

SREBF1c mRNA

SRELXRE

FASN, ACC, SCD1

Supplementary Figure S6. Proposed feedback mechanism to regulate SREBP-1 level by intronic miR-33b.

2b

2c 2d

4a 4b

5a

S2a

Supplementary Figure S7. Scans of the original blots in figure 2b, 2c, 2d, 4a, 4b, 5a and S2a.

200 kDABCA1

97 kD116 kD

55 kD

97 kD116 kD

55 kD

Precursor SREBP-1

short exposure

long exposure

MatureSREBP-1

AMPKα55 kD

97 kD

55 kD

97 kD

CPT1a

TF2B 36 kD

42 kDβ-actin

42 kDβ-actin

200 kD

116 kDSRC1

55 kDPCK1

36 kD31 kD

CREB

42 kDβ-actin

36 kD31 kD

G6Pase

200 kD

116 kD 55 kD

42 kD

β-actinABCG1ABCA1

10.7 kb

4.8 kb 6.6 kb

3000 bp

1000 bp500 bp

200 bp

3000 bp

1000 bp500 bp

6.6 kb

35.2 kb19.6 kb

35.2 kb19.6 kb 10.7 kb

4.8 kb