Supporting InformationYang et al. 10.1073/pnas.1605733113

GFP

Pol II

FRIELF

6:GFP/el

f6

Fig. S1. Pol II is not enriched by ELF6:GFP immunoprecipitation. Total protein was extracted from 14-d-old seedlings. GFP pull-down was performed byGFP-Trap. FRI was used as the control. 8WG16 was used to detect Pol II using Western blotting.

Yang et al. www.pnas.org/cgi/content/short/1605733113 1 of 6

SDG8:GFP/sd

g8

SDG8:GFP/el

f6 sd

g8

SD

G8

0

0.5

1.5

2.0

1.0

ELF6:G

FP/elf6

ELF6:G

FP/elf6

sdg8

0

0.1

0.2

0.3

ELF

6

A B

C D

SD

G8:

GFP

/elf6

sdg

8E

LF6:

GFP

/elf6

sdg

8

Fig. S2. SDG8 and ELF6 expression levels in different transgenic plants. (A) SDG8:GFP expression in SDG8:GFP/elf6 sdg8 in a root of a 9-d-old seedling. (B) SDG8expression levels in SDG8:GFP/sdg8 and SDG8:GFP/elf6 sdg8 transgenic plants. The value represents the ratio SDG8/UBC. Means ± SEM from three independentexperiments are shown. (C) ELF6:GFP expression in ELF6:GFP/elf6 sdg8 in a root of a 9-d-old seedling. (D) ELF6 expression levels in ELF6:GFP/elf6 and ELF6:GFP/elf6 sdg8 transgenic plants. The value represents the ratio of ELF6/UBC. Means ± SEM from three independent experiments are shown.

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FRIelf6

sdg8elf6 sdg8

Pol

II 8

WG

16 (I

nput

%)

0

1.2

0.9

0.6

0.3

-2000 0 2000 4000 6000 bp

FRIelf6sdg8elf6 sdg8

0

2.0

1.5

1.0

0.5

ACTIN STM AtSN1

A B

FRIelf6

sdg8elf6 sdg8

Tota

l RN

A fo

ld c

hang

e

0

15

5

10

20

1000 2000 3000 4000 bp0

FRIelf6

sdg8elf6 sdg8

Splice

d FLC

FLC ex

on1

Unspli

ced F

LC0

10

20

30

RN

A fo

ld c

hang

eP

ol II

8W

G16

(Inp

ut %

)C

D

Fig. S3. RNA fold changes and total Pol II levels at FLC. (A) Relative FLC RNA levels in FRI and the mutants. Spliced FLC (over exons 4–6), FLC exon 1, andunspliced FLC (unspliced introns 2 and 3) are shown. Levels are normalized to internal reference gene UBC, and then normalized to sdg8 (set as 1). (B) Total PolII levels at FLC in FRI and the mutants, as detected by 8WG16 antibody. (C) Pol II levels at actively transcribed gene ACTIN, silenced gene STM, and transposonelement AtSN1 were used as experimental controls for B. (D) Total RNA fold changes along FLC intron 1 in FRI and the mutants. Levels are normalized tointernal reference gene UBC, and then normalized to sdg8 (set as 1). In A–D, means ± SEM from three independent experiments are shown.

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ELF6 expression levels

SDG8 expression levels

SQ16

SQ17a

SQ17b1

SQ17b2

SQ18 NV

2WV T0

3WV T0

4WV T0

5WV T0

5WV T7

Silique development Vernalization

SQ16

SQ17a

SQ17b1

SQ17b2

SQ18 NV

2WV T0

3WV T0

4WV T0

5WV T0

5WV T7

Silique development Vernalization

0.18

0.15

0.12

0.09

0.06

0.03

0

ELF

6S

DG

8

0

1.2

0.9

0.6

0.3

A

B

Fig. S4. ELF6 and SDG8 expression levels in developing embryos and during vernalization. (A) ELF6 mRNA expression levels in developing embryos and duringvernalization. (B) SDG8 mRNA expression levels in developing embryos and during vernalization. The developing stages of siliques: immediately after fertil-ization, with petals still attached (SQ16); small and without petals (SQ17a); first (SQ17b1) and last (SQ17b2) mature green siliques; and yellow siliques (SQ18).The vernalization states are: nonvernalized plant materials (NV), plants after a certain period of cold exposure (e.g., 2WV T0, 2 wk of cold exposure), and plantsafter a 5-wk cold exposure followed by 7 d growth in the warm (5WV T7). The values represent the ratios FLC/UBC. Values are means ± SEM of three biologicalreplicates.

FLC

0

5

10

15

NV

2WV T0

3WV T0

4WV T0

5WV T0

5WV T7

5WV T20

FRIjmj13

elf6

elf6 jmj13

FLC expression levels in vernalization

Fig. S5. H3K27me3 demethylase mutants display similar FLC expression dynamics during/after cold exposure as FRI. Values are means ± SEM of three bi-ological replicates.

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-3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 7000 -3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 70000

0.4

0.8

1.2

0

0.4

0.8

1.2

-3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 70000

0.4

0.8

1.2

H3K

27m

e3H

3K27

me3

H3K

36m

e3

-3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 70000

0.4

0.8

1.2

H3K

36m

e3

0

0.4

0.8

1.2

H3K

27m

e3

0

0.4

0.8

1.2

H3K

36m

e3

-3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 7000 -3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 7000

FRI

elf6-3

FRI

elf6-3

FRI

elf6-3

FRI

elf6-3

FRI

elf6-3

FRI

elf6-3

H3K27me3 at 3WV T0

H3K27me3 at 3WV T4 H3K36me3 at 3WV T4

H3K27me3 at 3WV T10 H3K36me3 at 3WV T10

H3K36me3 at 3WV T0A B

C D

E F

* *

* *

*

*

*

*

*

** *

****

* *

***

** * *

Fig. S6. The H3K27me3 and H3K36me3 dynamics during/after cold exposure in elf6. (A to F) ChIP analysis histone modification levels. H3K27me3 levels in3WV T0 (A), 3WV T4 (C), 3WV T10 (E). STM was used as the internal control. Data are presented as the ratio of H3K27me3 FLC/H3 FLC to H3K27me3 STM/H3STM. H3K36me3 levels in 3WV T0 (B), 3WV T4 (D), and 3WV T10 (F) are shown. ACTIN was used as the internal control. Data are presented as the ratio ofH3K36me3 FLC/H3 FLC to H3K36me3 ACTIN/H3 ACTIN. Means ± SEM from three independent biological replicates are presented. Asterisk indicates significantdifferences between FRI and elf6, as determined by Student’s t test (P < 0.05).

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FRI ref6 FRIref

6R

oset

tle le

af n

umbe

r0

10

20

30

40B C

FRIref6

NV

2WV T0

3WV T0

4WV T0

5WV T0

5WV T7

5WV T21

5WV T28

15

12

9

6

3

0

FLC expression levels in vernalization

FLC

A

Fig. S7. ref6 displays late flowering after vernalization. (A) FLC expression levels during a time course of vernalization. FLC expression levels were measured innonvernalized plant materials (NV), plants after a certain period of cold exposure (e.g., 2WV T0, 2 wk of cold exposure), and plants after a 5-wk cold exposurewith a certain period of postcold growth (e.g., 5WV T7, 7 d warm growth after 5-wk cold exposure). Values are means ± SEM of three biological replicates. Thevalues represent the ratio FLC/UBC. (B) Flowering time of the plants after a 4-wk cold exposure, after which the plants were grown in long-day conditions.(C) Flowering time measured by rosette leaf number produced by the primary shoot apical meristem when the first flower was opening. The plants weretreated with a 4-wk cold exposure, then grown in long-day conditions. Values are means ± SEM from eight plants.

WT

elf6

jmj13

elf6 j

mj13

ref6

elf6 r

ef6

jmj13

ref6

elf6 j

mj13 re

f6

H3K27me3

H3

Fig. S8. H3K27me3 levels in H3K27 demethylase mutants. H3 was used as loading control.

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